Compounds useful as inhibitors of atr kinase

ABSTRACT

The present invention relates to pyridine compounds useful as inhibitors of ATR protein kinase. The invention also relates to pharmaceutically acceptable compositions comprising the compounds of this invention; methods of treating various diseases, disorders, and conditions using the compounds of this invention; processes for preparing the compounds of this invention; intermediates for the preparation of the compounds of this invention; and methods of using the compounds in in vitro applications, such as the study of kinases in biological and pathological phenomena; the study of intracellular signal transduction pathways mediated by such kinases; and the comparative evaluation of new kinase inhibitors. 
     The compounds of this invention have formula III: 
     
       
         
         
             
             
         
       
     
     wherein the variables are as defined herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This present application claims the benefit, under 35 U.S.C. §119, toU.S. Provisional Application No. 61/333,854, filed on May 12, 2010, theentire contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

ATR (“ATM and Rad3 related”) kinase is a protein kinase involved incellular responses to DNA damage. ATR kinase acts with ATM (“ataxiatelangiectasia mutated”) kinase and many other proteins to regulate acell's response to DNA damage, commonly referred to as the DNA DamageResponse (“DDR”). The DDR stimulates DNA repair, promotes survival andstalls cell cycle progression by activating cell cycle checkpoints,which provide time for repair. Without the DDR, cells are much moresensitive to DNA damage and readily die from DNA lesions induced byendogenous cellular processes such as DNA replication or exogenous DNAdamaging agents commonly used in cancer therapy.

Healthy cells can rely on a host of different proteins for DNA repairincluding the DDR kinase ATR. In some cases these proteins cancompensate for one another by activating functionally redundant DNArepair processes. On the contrary, many cancer cells harbour defects insome of their DNA repair processes, such as ATM signaling, and thereforedisplay a greater reliance on their remaining intact DNA repair proteinswhich include ATR.

In addition, many cancer cells express activated oncogenes or lack keytumour suppressors, and this can make these cancer cells prone todysregulated phases of DNA replication which in turn cause DNA damage.ATR has been implicated as a critical component of the DDR in responseto disrupted DNA replication. As a result, these cancer cells are moredependent on ATR activity for survival than healthy cells. Accordingly,ATR inhibitors may be useful for cancer treatment, either used alone orin combination with DNA damaging agents, because they shut down a DNArepair mechanism that is more important for cellular survival in manycancer cells than in healthy normal cells.

In fact, disruption of ATR function (e.g. by gene deletion) has beenshown to promote cancer cell death both in the absence and presence ofDNA damaging agents. This suggests that ATR inhibitors may be effectiveboth as single agents and as potent sensitizers to radiotherapy orgenotoxic chemotherapy.

ATR peptide can be expressed and isolated using a variety of methodsknown in the literature (see e.g., Ünsal-Kaçmaz et al, PNAS 99: 10, pp6673-6678, May 14, 2002; see also Kumagai et al. Cell 124, pp 943-955,Mar. 10, 2006; Unsal-Kacmaz et al. Molecular and Cellular Biology,February 2004, p 1292-1300; and Hall-Jackson et al. Oncogene 1999, 18,6707-6713).

For all of these reasons, there is a need for the development of potentand selective ATR inhibitors for the treatment of cancer, either assingle agents or as combination therapies with radiotherapy or genotoxicchemotherapy.

SUMMARY OF THE INVENTION

The present invention relates to pyridines compounds useful asinhibitors of ATR protein kinase. The invention also relates topharmaceutically acceptable compositions comprising the compounds ofthis invention; methods of treating various diseases, disorders, andconditions using the compounds of this invention; processes forpreparing the compounds of this invention; intermediates for thepreparation of the compounds of this invention; and methods of using thecompounds in in vitro applications, such as the study of kinases inbiological and pathological phenomena; the study of intracellular signaltransduction pathways mediated by such kinases; and the comparativeevaluation of new kinase inhibitors. These compounds have an unexpectedability to treat cancer as single agents. These compounds also showsurprising synergy with other cancer agents, such as cisplatin, incombination therapies.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of this invention provides a compound of Formula III

or a pharmaceutically acceptable salt thereof, wherein

-   L₂ is C₁₋₁₀ aliphatic chain where up to 3 methylene units of said    chain are optionally replaced with —O—, —S—, —N(R′)—, or —CO—;-   R′ is H or C₁₋₄alkyl;-   m is 0 or 1;-   R² is -Q or -Q-Q¹;-   Q is a 3-8 membered monocyclic ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; each Q is    independently and optionally substituted with 1-4 J^(Q) groups;-   Q is optionally fused to Q¹ to form a fused bicyclic ring Q-Q¹; or Q    and Q¹ are optionally joined together at a carbon atom to form a    spirocyclic bicyclic ring Q-Q¹; or Q and Q¹, taken together, form a    bridged bicyclic ring Q-Q¹ wherein said bridge is 1-3 atoms long;-   Q¹ is a 3-8 membered monocyclic ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; each Q¹ is    independently and optionally substituted with 1-4 J^(Q1) groups;-   L is —C(O)NH— or —C(O)N(C₁₋₆alkyl)-;-   n is 0 or 1;-   R¹ is a 5-6 membered monocyclic aryl or heteroaryl ring having 0-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur;    wherein said monocyclic aryl or heteroaryl ring is optionally fused    to another ring to form a 8-10 membered bicyclic aryl or heteroaryl    ring having 0-6 heteroatoms independently selected from nitrogen,    oxygen, or sulfur; R¹ is optionally substituted with 1-5 J¹ groups;-   each J^(Q), J^(Q1), and J¹ is independently halo, —CN, —NO₂, V—R, or    —(V²)_(m)-Q³;-   V is a C₁₋₁₀aliphatic chain wherein 0-3 methylene units are    optionally and independently replaced with oxygen, nitrogen, sulfur,    C(O), S(O), or S(O)₂; V is optionally substituted with 1-6    occurrences of J^(V);-   V² is a C₁₋₁₀aliphatic chain wherein 0-3 methylene units are    optionally and independently replaced with oxygen, nitrogen, sulfur,    C(O), S(O), or S(O)₂; V is optionally substituted with 1-6    occurrences of J^(V2);-   m is 0 or 1;-   Q³ is a 3-8 membered saturated or unsaturated monocyclic ring having    0-4 heteroatoms independently selected from nitrogen, oxygen, or    sulfur, or a 8-10 membered saturated or unsaturated bicyclic ring    having 0-6 heteroatoms independently selected from nitrogen, oxygen,    or sulfur; each Q³ is optionally substituted with 1-5 J^(Q3);-   each J^(V) and J^(V2) is independently halogen, CN, NH₂, NO₂,    C₁₋₄aliphatic, NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, OH,    O(C₁₋₄aliphatic), CO₂H, CO(C₁₋₄aliphatic), CO₂(C₁₋₄aliphatic),    C(O)NH₂, C(O)NH(C₁₋₄aliphatic),    C(O)N(C₁₋₄aliphatic)₂,NHCO(C₁₋₄aliphatic),    N(C₁₋₄aliphatic)CO(C₁₋₄aliphatic), SO₂(C₁₋₄aliphatic),    NHSO₂(C₁₋₄aliphatic), or N(C₁₋₄aliphatic)SO₂(C₁₋₄aliphatic), wherein    said C₁₋₄aliphatic is optionally substituted with halo;-   J^(Q3) is halo, oxo, CN, NO₂, X—R, or —(X)_(p)-Q⁴,-   p is 0 or 1;-   X is C₁₋₁₀aliphatic; wherein 1-3 methylene units of said    C₁₋₆aliphatic are optionally replaced with —NR, —O—, —S—, C(O),    S(O)₂, or S(O); wherein X is optionally and independently    substituted with 1-4 occurrences of NH₂, NH(C₁₋₄aliphatic),    N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄aliphatic),    NO₂, CN, CO(C₁₋₄aliphatic), CO₂H, CO₂(C₁₋₄aliphatic), C(O)NH₂,    C(O)NH(C₁₋₄aliphatic), C(O)N(C₁₋₄aliphatic)₂, SO(C₁₋₄aliphatic),    SO₂(C₁₋₄aliphatic), SO₂NH(C₁₋₄aliphatic), SO₂NH(C₁₋₄aliphatic),    NHC(O)(C₁₋₄aliphatic), N(C₁₋₄aliphatic)C(O)(C₁₋₄aliphatic),    NHSO₂(C₁₋₄aliphatic), or N(C₁₋₄aliphatic)SO₂(C₁₋₄aliphatic), wherein    said C₁₋₄aliphatic is optionally substituted with 1-3 occurrences of    halo;-   Q⁴ is a 3-8 membered saturated or unsaturated monocyclic ring having    0-4 heteroatoms independently selected from nitrogen, oxygen, or    sulfur, or a 8-10 membered saturated or unsaturated bicyclic ring    having 0-6 heteroatoms independently selected from nitrogen, oxygen,    or sulfur; each Q⁴ is optionally substituted with 1-5 J^(Q4);-   J^(Q4) is halo, CN, or C₁₋₄alkyl wherein up to 2 methylene units are    optionally replaced with O, N, S, C(O), S(O), or S(O)₂;-   R is H or C₁₋₄alkyl wherein said C₁₋₄alkyl is optionally substituted    with 1-4 halo.

Another aspect of this invention provides a compound of Formula III′

or a pharmaceutically acceptable salt thereof, wherein

-   L₂ is C₁₋₁₀ aliphatic chain where up to 3 methylene units of said    chain are optionally replaced with —O—, —S—, —N(R′)—, or —CO—;-   R′ is H or C₁₋₄alkyl;-   m is 0 or 1;-   R² is -Q or -Q-Q¹;-   Q is a 3-8 membered monocyclic ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; each Q is    independently and optionally substituted with 1-4 J^(Q) groups;-   Q is optionally fused to Q¹ to form a fused bicyclic ring Q-Q¹; or Q    and Q¹ are optionally joined together at a carbon atom to form a    spirocyclic bicyclic ring Q-Q¹; or Q and Q¹, taken together, form a    bridged bicyclic ring Q-Q¹ wherein said bridge is 1-3 atoms long;-   Q¹ is a 3-8 membered monocyclic ring having 0-4 heteroatoms    independently selected from nitrogen, oxygen, or sulfur; each Q¹ is    independently and optionally substituted with 1-4 J^(Q1) groups;-   L is —C(O)NH— or —C(O)N(C₁₋₆alkyl)-;-   n is 0 or 1;-   R¹ is a 5-6 membered monocyclic aryl or heteroaryl ring having 0-4    heteroatoms independently selected from nitrogen, oxygen, or sulfur;    wherein said monocyclic aryl or heteroaryl ring is optionally fused    to another ring to form a 8-10 membered bicyclic aryl or heteroaryl    ring having 0-6 heteroatoms independently selected from nitrogen,    oxygen, or sulfur; R¹ is optionally substituted with 1-5 J¹ groups;-   each J^(Q), J^(Q1), J¹, and J² is independently halo, —CN, —NO₂,    V—R, or —(V²)_(m)-Q³;-   V is a C₁₋₁₀aliphatic chain wherein 0-3 methylene units are    optionally and independently replaced with oxygen, nitrogen, sulfur,    C(O), S(O), or S(O)₂; V is optionally substituted with 1-6    occurrences of J^(V);-   V² is a C₁₋₁₀aliphatic chain wherein 0-3 methylene units are    optionally and independently replaced with oxygen, nitrogen, sulfur,    C(O), S(O), or S(O)₂; V is optionally substituted with 1-6    occurrences of J^(V2);-   m is 0 or 1;-   Q³ is a 3-8 membered saturated or unsaturated monocyclic ring having    0-4 heteroatoms independently selected from nitrogen, oxygen, or    sulfur, or a 8-10 membered saturated or unsaturated bicyclic ring    having 0-6 heteroatoms independently selected from nitrogen, oxygen,    or sulfur; each Q³ is optionally substituted with 1-5 J^(Q3);-   each J^(V) and J^(V2) is independently halogen, CN, NH₂, NO₂,    NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, OH, O(C₁₋₄aliphatic), CO₂H,    CO(C₁₋₄aliphatic), CO₂(C₁₋₄aliphatic), C(O)NH₂,    C(O)NH(C₁₋₄aliphatic), C(O)N(C₁₋₄aliphatic)₂,NHCO(C₁₋₄aliphatic),    N(C₁₋₄aliphatic)CO(C₁₋₄aliphatic), SO₂(C₁₋₄aliphatic),    NHSO₂(C₁₋₄aliphatic), or N(C₁₋₄aliphatic)SO₂(C₁₋₄aliphatic), wherein    said C₁₋₄aliphatic is optionally substituted with halo;-   J^(Q3) is halo, oxo, CN, NO₂, X—R, or —(X)_(p)-Q⁴,-   p is 0 or 1;-   X is C₁₋₁₀aliphatic; wherein 1-3 methylene units of said    C₁₋₆aliphatic are optionally replaced with —NR, —O—, —S—, C(O),    S(O)₂, or S(O); wherein X is optionally and independently    substituted with 1-4 occurrences of NH₂, NH(C₁₋₄aliphatic),    N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH, O(C₁₋₄aliphatic),    NO₂, CN, CO(C₁₋₄aliphatic), CO₂H, CO₂(C₁₋₄aliphatic), C(O)NH₂,    C(O)NH(C₁₋₄aliphatic), C(O)N(C₁₋₄aliphatic)₂, SO(C₁₋₄aliphatic),    SO₂(C₁₋₄aliphatic), SO₂NH(C₁₋₄aliphatic), SO₂NH(C₁₋₄aliphatic),    NHC(O)(C₁₋₄aliphatic), N(C₁₋₄aliphatic)C(O)(C₁₋₄aliphatic),    NHSO₂(C₁₋₄aliphatic), or N(C₁₋₄aliphatic)SO₂(C₁₋₄aliphatic), wherein    said C₁₋₄aliphatic is optionally substituted with 1-3 occurrences of    halo;-   Q⁴ is a 3-8 membered saturated or unsaturated monocyclic ring having    0-4 heteroatoms independently selected from nitrogen, oxygen, or    sulfur, or a 8-10 membered saturated or unsaturated bicyclic ring    having 0-6 heteroatoms independently selected from nitrogen, oxygen,    or sulfur; each Q⁴ is optionally substituted with 1-5 J^(Q4);-   J^(Q4) is halo, CN, or C₁₋₄alkyl wherein up to 2 methylene units are    optionally replaced with O, N, S, C(O), S(O), or S(O)₂;-   R is H or C₁₋₄alkyl wherein said C₁₋₄alkyl is optionally substituted    with 1-4 halo.

In some embodiments m is 0.

In other embodiments, n is 0. In some embodiments R¹ is isoxazolyl oroxadiazolyl. According to some embodiments, R¹ is optionally substitutedwith one occurrence of J¹. In some embodiments, J¹ is —(V²)_(m)-Q³wherein m is 0. In other embodiments, J¹ is phenyl. In yet otherembodiments, J^(Q3) is halo or X—R. In some embodiment, J¹ is phenyloptionally substituted with CH₂NHCH₃, methyl, or fluoro.

In other embodiments, n is 1. In some embodiments, L is —C(O)NH— and R¹is phenyl.

According to another embodiment, Q is aromatic. In some embodiments, R²is phenyl, pyridinyl, pyrimidinyl, pyrazinyl, or thienyl. In otherembodiment, R² is pyridinyl.

In some embodiments, Q is optionally substituted with one occurrence ofJ^(Q). In some embodiments, J^(Q) is SO₂(C₁₋₆alkyl), SO₂(furanyl), orCO(homopiperazinyl).

According to another embodiment, Q is nonaromatic. In some embodiments,R² is 1,2,3,6-tetrahydropyridinyl or N-alkyl pyridone.

According to another embodiment, J² is V—R or —(V²)_(m)-Q³. In someembodiments, V is S(O)₂ or C(O); and V² is S(O)₂ or C(O). In someembodiments, R is C₁₋₄alkyl and Q³ is a 3-7 membered saturated orunsaturated monocyclic ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

According to another embodiment, J^(Q) is V—R or —(V²)_(m)-Q³. In someembodiments, V is S(O)₂ or C(O); and V² is S(O)₂ or C(O). In someembodiments, R is C₁₋₄alkyl and Q³ is a 3-7 membered saturated orunsaturated monocyclic ring having 0-2 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur.

According to some embodiments, R² is optionally substituted with J².

According to another embodiment, n is 0 or 1; L is —C(O)NH—; R¹ isphenyl, oxadiazolyl, or isoxazolyl; m is 0; R² is phenyl, pyridinyl,pyrimidinyl, pyrazinyl, thienyl, or 1,2,3,6-tetrahydropyridinyl; and J²is V—R or —(V²)_(m)-Q³ wherein V is SO₂ or CO, R is C₁₋₄alkyl; V² isS(O)₂ or C(O); and Q³ is a 5-7 membered monocyclic ring having 0-2heteroatoms selected from oxygen, nitrogen, or sulfur.

According to another embodiment, n is 0 or 1; L is —C(O)NH—; R¹ isphenyl, oxadiazolyl, or isoxazolyl; m is 0; R² is phenyl, pyridinyl,pyrimidinyl, pyrazinyl, thienyl, or 1,2,3,6-tetrahydropyridinyl; andJ^(Q) is V—R or —(V²)_(m)-Q³ wherein V is SO₂ or CO, R is C₁₋₄alkyl; V²is S(O)₂ or C(O); and Q³ is a 5-7 membered monocyclic ring having 0-2heteroatoms selected from oxygen, nitrogen, or sulfur.

According to another embodiment, n is 0 or 1; L is —C(O)NH—; R¹ isphenyl, oxadiazolyl, or isoxazolyl; J¹ is phenyl optionally substitutedwith CH₂NHCH₃, methyl, or fluoro; m is 0; R² is phenyl, pyridinyl,pyrazinyl, thienyl, or 1,2,3,6-tetrahydropyridinyl; and J² isSO₂(furanyl), CO(homopiperazinyl), or SO₂(C₁₋₆alkyl).

Another embodiment provides a compound from the following Table:

TABLE III

III-1

III-2

III-3

III-4

III-5

III-6

III-7

III-8

III-9

III-10

III-11

III-12

In some embodiments, the variables are as depicted in the compounds ofthe disclosure including compounds in the tables above.

Compounds of this invention include those described generally herein,and are further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As described herein, a specified number range of atoms includes anyinteger therein. For example, a group having from 1-4 atoms could have1, 2, 3, or 4 atoms.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally herein, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds.

Unless otherwise indicated, a substituent connected by a bond drawn fromthe center of a ring means that the substituent can be bonded to anyposition in the ring. In example i below, for instance, J¹ can be bondedto any position on the pyridyl ring. For bicyclic rings, a bond drawnthrough both rings indicates that the substituent can be bonded from anyposition of the bicyclic ring. In example 11 below, for instance, J¹ canbe bonded to the 5-membered ring (on the nitrogen atom, for instance),and to the 6-membered ring.

The term “stable”, as used herein, refers to compounds that are notsubstantially altered when subjected to conditions to allow for theirproduction, detection, recovery, purification, and use for one or moreof the purposes disclosed herein. In some embodiments, a stable compoundor chemically feasible compound is one that is not substantially alteredwhen kept at a temperature of 40° C. or less, in the absence of moistureor other chemically reactive conditions, for at least a week.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched), branched, or cyclic, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation that has a single point ofattachment to the rest of the molecule.

Unless otherwise specified, aliphatic groups contain 1-20 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-10aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-8 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-6 aliphatic carbon atoms, and in yet other embodimentsaliphatic groups contain 1-4 aliphatic carbon atoms. Aliphatic groupsmay be linear or branched, substituted or unsubstituted alkyl, alkenyl,or alkynyl groups. Specific examples include, but are not limited to,methyl, ethyl, isopropyl, n-propyl, sec-butyl, vinyl, n-butenyl,ethynyl, and tert-butyl. Aliphatic groups may also be cyclic, or have acombination of linear or branched and cyclic groups. Examples of suchtypes of aliphatic groups include, but are not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, —CH₂-cyclopropyl,CH₂CH₂CH(CH₃)-cyclohexyl.

The term “cycloaliphatic” (or “carbocycle” or “carbocyclyl”) refers to amonocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule wherein any individual ring in said bicyclic ringsystem has 3-7 members. Examples of cycloaliphatic groups include, butare not limited to, cycloalkyl and cycloalkenyl groups. Specificexamples include, but are not limited to, cyclohexyl, cyclopropenyl, andcyclobutyl.

The term “heterocycle”, “heterocyclyl”, or “heterocyclic” as used hereinmeans non-aromatic, monocyclic, bicyclic, or tricyclic ring systems inwhich one or more ring members are an independently selected heteroatom.In some embodiments, the “heterocycle”, “heterocyclyl”, or“heterocyclic” group has three to fourteen ring members in which one ormore ring members is a heteroatom independently selected from oxygen,sulfur, nitrogen, or phosphorus, and each ring in the system contains 3to 7 ring members.

Examples of heterocycles include, but are not limited to,3-1H-benzimidazol-2-one, 3-(1-alkyl)-benzimidazol-2-one,2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl,3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino,2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl,2-pyrrolidinyl, 3-pyrrolidinyl, 1-tetrahydropiperazinyl,2-tetrahydropiperazinyl, 3-tetrahydropiperazinyl, 1-piperidinyl,2-piperidinyl, 3-piperidinyl, 1-pyrazolinyl, 3-pyrazolinyl,4-pyrazolinyl, 5-pyrazolinyl, 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl,4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl,5-imidazolidinyl, indolinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, benzothiolane, benzodithiane, and1,3-dihydro-imidazol-2-one.

Cyclic groups, (e.g. cycloaliphatic and heterocycles), can be linearlyfused, bridged, or spirocyclic.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation. As would be known by one of skill in theart, unsaturated groups can be partially unsaturated or fullyunsaturated. Examples of partially unsaturated groups include, but arenot limited to, butene, cyclohexene, and tetrahydropyridine. Fullyunsaturated groups can be aromatic, anti-aromatic, or non-aromatic.Examples of fully unsaturated groups include, but are not limited to,phenyl, cyclooctatetraene, pyridyl, thienyl, and1-methylpyridin-2(1H)-one.

The term “alkoxy”, or “thioalkyl”, as used herein, refers to an alkylgroup, as previously defined, attached through an oxygen (“alkoxy”) orsulfur (“thioalkyl”) atom.

The terms “haloalkyl”, “haloalkenyl”, “haloaliphatic”, and “haloalkoxy”mean alkyl, alkenyl or alkoxy, as the case may be, substituted with oneor more halogen atoms. This term includes perfluorinated alkyl groups,such as —CF₃ and —CF₂CF₃.

The terms “halogen”, “halo”, and “hal” mean F, Cl, Br, or I. The term“aryl” used alone or as part of a larger moiety as in “aralkyl”,“aralkoxy”, or “aryloxyalkyl”, refers to monocyclic, bicyclic, andtricyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. The term “aryl” may beused interchangeably with the term “aryl ring”.

The term “heteroaryl”, used alone or as part of a larger moiety as in“heteroaralkyl” or “heteroarylalkoxy”, refers to monocyclic, bicyclic,and tricyclic ring systems having a total of five to fourteen ringmembers, wherein at least one ring in the system is aromatic, at leastone ring in the system contains one or more heteroatoms, and whereineach ring in the system contains 3 to 7 ring members. The term“heteroaryl” may be used interchangeably with the term “heteroaryl ring”or the term “heteroaromatic”. Examples of heteroaryl rings include, butare not limited to, 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl,4-imidazolyl, 5-imidazolyl, benzimidazolyl, 3-isoxazolyl, 4-isoxazolyl,5-isoxazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, N-pyrrolyl,2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g., 3-pyridazinyl),2-thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (e.g., 5-tetrazolyl),triazolyl (e.g., 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl,benzofuryl, benzothiophenyl, indolyl (e.g., 2-indolyl), pyrazolyl (e.g.,2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,3-triazolyl, 1,2,3-thiadiazolyl,1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, purinyl, pyrazinyl,1,3,5-triazinyl, quinolinyl (e.g., 2-quinolinyl, 3-quinolinyl,4-quinolinyl), and isoquinolinyl (e.g., 1-isoquinolinyl,3-isoquinolinyl, or 4-isoquinolinyl).

It shall be understood that the term “heteroaryl” includes certain typesof heteroaryl rings that exist in equilibrium between two differentforms. More specifically, for example, species such hydropyridine andpyridinone (and likewise hydroxypyrimidine and pyrimidinone) are meantto be encompassed within the definition of “heteroaryl.”

The term “protecting group” and “protective group” as used herein, areinterchangeable and refer to an agent used to temporarily block one ormore desired functional groups in a compound with multiple reactivesites. In certain embodiments, a protecting group has one or more, orpreferably all, of the following characteristics: a) is addedselectively to a functional group in good yield to give a protectedsubstrate that is b) stable to reactions occurring at one or more of theother reactive sites; and c) is selectively removable in good yield byreagents that do not attack the regenerated, deprotected functionalgroup. As would be understood by one skilled in the art, in some cases,the reagents do not attack other reactive groups in the compound. Inother cases, the reagents may also react with other reactive groups inthe compound. Examples of protecting groups are detailed in Greene, T.W., Wuts, P. G in “Protective Groups in Organic Synthesis”, ThirdEdition, John Wiley & Sons, New York: 1999 (and other editions of thebook), the entire contents of which are hereby incorporated byreference. The term “nitrogen protecting group”, as used herein, refersto an agent used to temporarily block one or more desired nitrogenreactive sites in a multifunctional compound. Preferred nitrogenprotecting groups also possess the characteristics exemplified for aprotecting group above, and certain exemplary nitrogen protecting groupsare also detailed in Chapter 7 in Greene, T. W., Wuts, P. G in“Protective Groups in Organic Synthesis”, Third Edition, John Wiley &Sons, New York: 1999, the entire contents of which are herebyincorporated by reference.

In some embodiments, a methylene unit of an alkyl or aliphatic chain isoptionally replaced with another atom or group. Examples of such atomsor groups include, but are not limited to, nitrogen, oxygen, sulfur,—C(O)—, —C(═N—CN)—, —C(═NR)—, —C(═NOR)—, —SO—, and —SO₂—. These atoms orgroups can be combined to form larger groups. Examples of such largergroups include, but are not limited to, —OC(O)—, —C(O)CO—, —CO₂—,—C(O)NR—, —C(═N—CN), —NRCO—, —NRC(O)O—, —SO₂NR—, —NRSO₂—, —NRC(O)NR—,—OC(O)NR—, and —NRSO₂NR—, wherein R is, for example, H or C₁₋₆aliphatic.It should be understood that these groups can be bonded to the methyleneunits of the aliphatic chain via single, double, or triple bonds. Anexample of an optional replacement (nitrogen atom in this case) that isbonded to the aliphatic chain via a double bond would be —CH₂CH═N—CH₃.In some cases, especially on the terminal end, an optional replacementcan be bonded to the aliphatic group via a triple bond. One example ofthis would be CH₂CH₂CH₂C≡N. It should be understood that in thissituation, the terminal nitrogen is not bonded to another atom.

It should also be understood that, the term “methylene unit” can alsorefer to branched or substituted methylene units. For example, in anisopropyl moiety [—CH(CH₃)₂], a nitrogen atom (e.g. NR) replacing thefirst recited “methylene unit” would result in dimethylamine [—N(CH₃)₂].In instances such as these, one of skill in the art would understandthat the nitrogen atom will not have any additional atoms bonded to it,and the “R” from “NR” would be absent in this case.

Unless otherwise indicated, the optional replacements form a chemicallystable compound. Optional replacements can occur both within the chainand/or at either end of the chain; i.e. both at the point of attachmentand/or also at the terminal end. Two optional replacements can also beadjacent to each other within a chain so long as it results in achemically stable compound. For example, a C₃ aliphatic can beoptionally replaced by 2 nitrogen atoms to form —C—N≡N. The optionalreplacements can also completely replace all of the carbon atoms in achain. For example, a C₃ aliphatic can be optionally replaced by —NR—,—C(O)—, and —NR— to form —NRC(O)NR— (a urea).

Unless otherwise indicated, if the replacement occurs at the terminalend, the replacement atom is bound to a hydrogen atom on the terminalend. For example, if a methylene unit of —CH₂CH₂CH₃ were optionallyreplaced with —O—, the resulting compound could be —OCH₂CH₃, —CH₂OCH₃,or —CH₂CH₂OH. It should be understood that if the terminal atom does notcontain any free valence electrons, then a hydrogen atom is not requiredat the terminal end (e.g., —CH₂CH₂CH═O or —CH₂CH₂C≡N).

Unless otherwise indicated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, geometric,conformational, and rotational) forms of the structure. For example, theR and S configurations for each asymmetric center, (Z) and (E) doublebond isomers, and (Z) and (E) conformational isomers are included inthis invention. As would be understood to one skilled in the art, asubstituent can freely rotate around any rotatable bonds. For example, asubstituent drawn as

also represents

Therefore, single stereochemical isomers as well as enantiomeric,diastereomeric, geometric, conformational, and rotational mixtures ofthe present compounds are within the scope of the invention.

Unless otherwise indicated, all tautomeric forms of the compounds of theinvention are within the scope of the invention.

Additionally, unless otherwise indicated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures except for the replacement of hydrogen by deuteriumor tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enrichedcarbon are within the scope of this invention. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

Pharmaceutically Acceptable Salts

The compounds of this invention can exist in free form for treatment, orwhere appropriate, as a pharmaceutically acceptable salt.

A “pharmaceutically acceptable salt” means any non-toxic salt of acompound of this invention that, upon administration to a recipient, iscapable of providing, either directly or indirectly, a compound of thisinvention or an inhibitorily active metabolite or residue thereof. Asused herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof the ATR protein kinase.

Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporatedherein by reference. Pharmaceutically acceptable salts of the compoundsof this invention include those derived from suitable inorganic andorganic acids and bases. These salts can be prepared in situ during thefinal isolation and purification of the compounds. Acid addition saltscan be prepared by 1) reacting the purified compound in its free-basedform with a suitable organic or inorganic acid and 2) isolating the saltthus formed.

Examples of pharmaceutically acceptable, nontoxic acid addition saltsare salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, glycolate, gluconate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate,lauryl sulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like.

Base addition salts can be prepared by 1) reacting the purified compoundin its acid form with a suitable organic or inorganic base and 2)isolating the salt thus formed. Salts derived from appropriate basesinclude alkali metal (e.g., sodium, lithium, and potassium), alkalineearth metal (e.g., magnesium and calcium), ammonium and N⁺(C₁₋₄alkyl)₄salts. This invention also envisions the quaternization of any basicnitrogen-containing groups of the compounds disclosed herein. Water oroil-soluble or dispersible products may be obtained by suchquaternization.

Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate. Other acids and bases,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acid orbase addition salts.

ABBREVIATIONS

The following abbreviations are used:

-   DMSO dimethyl sulfoxide-   ATP adenosine triphosphate-   ¹HNMR proton nuclear magnetic resonance-   HPLC high performance liquid chromatography-   LCMS liquid chromatography-mass spectrometry-   TLC thin layer chromatography-   Rt retention time

Compound Uses

One aspect of this invention provides compounds that are inhibitors ofATR kinase, and thus are useful for treating or lessening the severityof a disease, condition, or disorder where ATR is implicated in thedisease, condition, or disorder.

Another aspect of this invention provides compounds that are useful forthe treatment of diseases, disorders, and conditions characterized byexcessive or abnormal cell proliferation. Such diseases include, aproliferative or hyperproliferative disease. Examples of proliferativeand hyperproliferative diseases include, without limitation, cancer andmyeloproliferative disorders.

In some embodiments, said compounds are selected from the groupconsisting of a compound of formula III. Te term “cancer” includes, butis not limited to the following cancers. Oral: buccal cavity, lip,tongue, mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: bronchogenic carcinoma (squamous cell or epidermoid,undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, larynx,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or smallintestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi'ssarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), largebowel or large intestines (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal; rectum,Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma, leukemia), bladder and urethra (squamouscell carcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast;Hematologic: blood (myeloid leukemia [acute and chronic], acutelymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferativediseases, multiple myeloma, myelodysplastic syndrome), Hodgkin'sdisease, non-Hodgkin's lymphoma [malignant lymphoma] hairy cell;lymphoid disorders; Skin: malignant melanoma, basal cell carcinoma,squamous cell carcinoma, Karposi's sarcoma, keratoacanthoma, molesdysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis,Thyroid gland: papillary thyroid carcinoma, follicular thyroidcarcinoma; undifferentiated thyroid cancer; medullary thyroid carcinoma,multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type2B, familial medullary thyroid cancer, pheochromocytoma, paraganglioma;and Adrenal glands: neuroblastoma.

Thus, the term “cancerous cell” as provided herein, includes a cellafflicted by any one of the above-identified conditions. In someembodiments, the cancer is selected from colorectal, thyroid, or breastcancer.

The term “myeloproliferative disorders”, includes disorders such aspolycythemia vera, thrombocythemia, myeloid metaplasia withmyelofibrosis, hypereosinophilic syndrome, juvenile myelomonocyticleukemia, systemic mast cell disease, and hematopoietic disorders, inparticular, acute-myelogenous leukemia (AML), chronic-myelogenousleukemia (CML), acute-promyelocytic leukemia (APL), and acutelymphocytic leukemia (ALL).

Pharmaceutically Acceptable Derivatives or Prodrugs

In addition to the compounds of this invention, pharmaceuticallyacceptable derivatives or prodrugs of the compounds of this inventionmay also be employed in compositions to treat or prevent the hereinidentified disorders.

The compounds of this invention can also exist as pharmaceuticallyacceptable derivatives.

A “pharmaceutically acceptable derivative” is an adduct or derivativewhich, upon administration to a patient in need, is capable ofproviding, directly or indirectly, a compound as otherwise describedherein, or a metabolite or residue thereof. Examples of pharmaceuticallyacceptable derivatives include, but are not limited to, esters and saltsof such esters.

A “pharmaceutically acceptable derivative or prodrug” means anypharmaceutically acceptable ester, salt of an ester or other derivativeor salt thereof of a compound, of this invention which, uponadministration to a recipient, is capable of providing, either directlyor indirectly, a compound of this invention or an inhibitorily activemetabolite or residue thereof. Particularly favoured derivatives orprodrugs are those that increase the bioavailability of the compounds ofthis invention when such compounds are administered to a patient (e.g.,by allowing an orally administered compound to be more readily absorbedinto the blood) or which enhance delivery of the parent compound to abiological compartment (e.g., the brain or lymphatic system) relative tothe parent species.

Pharmaceutically acceptable prodrugs of the compounds of this inventioninclude, without limitation, esters, amino acid esters, phosphateesters, metal salts and sulfonate esters.

Pharmaceutical Compositions

The present invention also provides compounds and compositions that areuseful as inhibitors of ATR kinase.

One aspect of this invention provides pharmaceutically acceptablecompositions that comprise any of the compounds as described herein, andoptionally comprise a pharmaceutically acceptable carrier, adjuvant orvehicle.

The pharmaceutically acceptable carrier, adjuvant, or vehicle, as usedherein, includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W.Martin (Mack Publishing Co., Easton, Pa., 1980) discloses variouscarriers used in formulating pharmaceutically acceptable compositionsand known techniques for the preparation thereof. Except insofar as anyconventional carrier medium is incompatible with the compounds of theinvention, such as by producing any undesirable biological effect orotherwise interacting in a deleterious manner with any othercomponent(s) of the pharmaceutically acceptable composition, its use iscontemplated to be within the scope of this invention.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, glycine, sorbicacid, or potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, wool fat, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol or polyethylene glycol; esters such as ethyl oleate and ethyllaurate; agar; buffering agents such as magnesium hydroxide and aluminumhydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer'ssolution; ethyl alcohol, and phosphate buffer solutions, as well asother non-toxic compatible lubricants such as sodium lauryl sulfate andmagnesium stearate, as well as coloring agents, releasing agents,coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Combination Therapies

Another aspect of this invention is directed towards a method oftreating cancer in a subject in need thereof, comprising administrationof a compound of this invention or a pharmaceutically acceptable saltthereof, and an additional therapeutic agent. In some embodiments, saidmethod comprises the sequential or co-administration of the compound ora pharmaceutically acceptable salt thereof, and the additionaltherapeutic agent.

In some embodiments, said additional therapeutic agent is an anti-canceragent. In other embodiments, said additional therapeutic agent is aDNA-damaging agent. In yet other embodiments, said additionaltherapeutic agent is selected from radiation therapy, chemotherapy, orother agents typically used in combination with radiation therapy orchemotherapy, such as radiosensitizers and chemosensitizers.

As would be known by one of skill in the art, radiosensitizers areagents that can be used in combination with radiation therapy.Radiosensitizers work in various different ways, including, but notlimited to, making cancer cells more sensitive to radiation therapy,working in synergy with radiation therapy to provide an improvedsynergistic effect, acting additively with radiation therapy, orprotecting surrounding healthy cells from damage caused by radiationtherapy. Likewise chemosensitizers are agents that can be used incombination with chemotherapy. Similarly, chemosensitizers work invarious different ways, including, but not limited to, making cancercells more sensitive to chemotherapy, working in synergy withchemotherapy to provide an improved synergistic effect, actingadditively to chemotherapy, or protecting surrounding healthy cells fromdamage caused by chemotherapy.

Examples of DNA-damaging agents that may be used in combination withcompounds of this invention include, but are not limited to Platinatingagents, such as Carboplatin, Nedaplatin, Satraplatin and otherderivatives; Topo I inhibitors, such as Topotecan, irinotecan/SN38,rubitecan and other derivatives; Antimetabolites, such as Folic family(Methotrexate, Pemetrexed and relatives); Purine antagonists andPyrimidine antagonists (Thioguanine, Fludarabine, Cladribine,Cytarabine, Gemcitabine, 6-Mercaptopurine, 5-Fluorouracil (5FU) andrelatives); Alkylating agents, such as Nitrogen mustards(Cyclophosphamide, Melphalan, Chlorambucil, mechlorethamine, Ifosfamideand relatives); nitrosoureas (eg Carmustine); Triazenes (Dacarbazine,temozolomide); Alkyl sulphonates (eg Busulfan); Procarbazine andAziridines; Antibiotics, such as Hydroxyurea, Anthracyclines(doxorubicin, daunorubicin, epirubicin and other derivatives);Anthracenediones (Mitoxantrone and relatives); Streptomyces family(Bleomycin, Mitomycin C, actinomycin); and Ultraviolet light.

Other therapies or anticancer agents that may be used in combinationwith the inventive agents of the present invention include surgery,radiotherapy (in but a few examples, gamma-radiation, neutron beamradiotherapy, electron beam radiotherapy, proton therapy, brachytherapy,and systemic radioactive isotopes, to name a few), endocrine therapy,biologic response modifiers (interferons, interleukins, and tumornecrosis factor (TNF) to name a few), hyperthermia and cryotherapy,agents to attenuate any adverse effects (e.g., antiemetics), and otherapproved chemotherapeutic drugs, including, but not limited to, the DNAdamaging agents listed herein, spindle poisons (Vinblastine,Vincristine, Vinorelbine, Paclitaxel), podophyllotoxins (Etoposide,Irinotecan, Topotecan), nitrosoureas (Carmustine, Lomustine), inorganicions (Cisplatin, Carboplatin), enzymes (Asparaginase), and hormones(Tamoxifen, Leuprolide, Flutamide, and Megestrol), Gleevec™, adriamycin,dexamethasone, and cyclophosphamide.

A compound of the instant invention may also be useful for treatingcancer in combination with any of the following therapeutic agents:abarelix (Plenaxis Depot®); aldesleukin (Prokine®); Aldesleukin(Proleukin®); Alemtuzumabb (Campath®); alitretinoin (Panretin®);allopurinol (Zyloprim®); altretamine (Hexylen®); amifostine (Ethyol®);anastrozole (Arimidex®); arsenic trioxide (Trisenox®); asparaginase(Elspar®); azacitidine (Vidaza®); bevacuzimab (Avastin®); bexarotenecapsules (Targretin®); bexarotene gel (Targretin®); bleomycin(Blenoxane®); bortezomib (Velcade®); busulfan intravenous (Busulfex®);busulfan oral (Myleran®); calusterone (Methosarb®); capecitabine(Xeloda®); carboplatin (Paraplatin®); carmustine (BCNU®, BiCNU®);carmustine (Gliadel®); carmustine with Polifeprosan 20 Implant (GliadelWafer®); celecoxib (Celebrex®); cetuximab (Erbitux®); chlorambucil(Leukeran®); cisplatin (Platinol®); cladribine (Leustatin®, 2-CdAe);clofarabine (Clolar®); cyclophosphamide (Cytoxan®, Neosar®);cyclophosphamide (Cytoxan Injection®); cyclophosphamide (CytoxanTablet®); cytarabine (Cytosar-U®); cytarabine liposomal (DepoCyt®);dacarbazine (DTIC-Dome®); dactinomycin, actinomycin D (Cosmegen®);Darbepoetin alfa (Aranesp®); daunorubicin liposomal (DanuoXome®);daunorubicin, daunomycin (Daunorubicin®); daunorubicin, daunomycin(Cerubidine®); Denileukin diftitox (Ontak®); dexrazoxane (Zinecard®);docetaxel (Taxotere®); doxorubicin (Adriamycin PFS®); doxorubicin(Adriamycin®, Rubex®); doxorubicin (Adriamycin PFS Injection®);doxorubicin liposomal (Doxil®); dromostanolone propionate(Dromostanolone®); dromostanolone propionate (masterone Injection®);Elliott's B Solution (Elliott's B Solution®); epirubicin (Ellence®);Epoetin alfa (Epogen®); erlotinib (Tarceva®); estramustine (Emcyt®);etoposide phosphate (Etopophos®); etoposide, VP-16 (Vepesid®);exemestane (Aromasin®); Filgrastim (Neupogen®); floxuridine(intraarterial) (FUDR®); fludarabine (Fludara®); fluorouracil, 5-FU(Adrucil®); fulvestrant (Faslodex®); gefitinib (Iressa®); gemcitabine(Gemzar®); gemtuzumab ozogamicin (Mylotarg®); goserelin acetate (ZoladexImplant®); goserelin acetate (Zoladex®); histrelin acetate (HistrelinImplant®); hydroxyurea (Hydrea®); Ibritumomab Tiuxetan (Zevalin®);idarubicin (Idamycin®); ifosfamide (IFEX®); imatinib mesylate(Gleevec®); interferon alfa 2a (Roferon A®); Interferon alfa-2b (IntronA®); irinotecan (Camptosar®); lenalidomide (Revlimid®); letrozole(Femara®); leucovorin (Wellcovorin®, Leucovorin®); Leuprolide Acetate(Eligard®); levamisole (Ergamisol®); lomustine, CCNU (CeeBU®);meclorethamine, nitrogen mustard (Mustargen®); megestrol acetate(Megace®); melphalan, L-PAM (Alkeran®); mercaptopurine, 6-MP(Purinethol®); mesna (Mesnex®); mesna (Mesnex Tabs®); methotrexate(Methotrexate®); methoxsalen (Uvadex®); mitomycin C (Mutamycin®);mitotane (Lysodren®); mitoxantrone (Novantrone®); nandrolonephenpropionate (Durabolin-50®); nelarabine (Arranon®); Nofetumomab(Verluma®); Oprelvekin (Neumega®); oxaliplatin (Eloxatin®); paclitaxel(Paxene®); paclitaxel (Taxol®); paclitaxel protein-bound particles(Abraxane®); palifermin (Kepivance®); pamidronate (Aredia®); pegademase(Adagen (Pegademase Bovine)®); pegaspargase (Oncaspar®); Pegfilgrastim(Neulasta®); pemetrexed disodium (Alimta®); pentostatin (Nipente);pipobroman (Vercyte®); plicamycin, mithramycin (Mithracin®); porfimersodium (Photofrin®); procarbazine (Matulane®); quinacrine (Atabrine®);Rasburicase (Elitek®); Rituximab (Rituxan®); sargramostim (Leukine®);Sargramostim (Prokine®); sorafenib (Nexavar®); streptozocin (Zanosar®);sunitinib maleate (Sutent®); talc (Sclerosol®); tamoxifen (Nolvadex®);temozolomide (Temodar®); teniposide, VM-26 (Vumon®); testolactone(Teslac®); thioguanine, 6-TG (Thioguanine®); thiotepa (Thioplex®);topotecan (Hycamtin®); toremifene (Fareston®); Tositumomab (Bexxar®);Tositumomab/1-131 tositumomab (Bexxar®); Trastuzumab (Herceptin®);tretinoin, ATRA (Vesanoid®); Uracil Mustard (Uracil Mustard Capsules®);valrubicin (Valstar®); vinblastine (Velban®); vincristine (Oncovin®);vinorelbine (Navelbine®); zoledronate (Zometa®) and vorinostat(Zolinza®).

For a comprehensive discussion of updated cancer therapies see,http://www.nci.nih.gov/, a list of the FDA approved oncology drugs athttp://www.fda.gov/cder/cancer/druglistframe.htm, and The Merck Manual,Seventeenth Ed. 1999, the entire contents of which are herebyincorporated by reference.

Compositions for Administration into a Subject

The ATR kinase inhibitors or pharmaceutical salts thereof may beformulated into pharmaceutical compositions for administration toanimals or humans. These pharmaceutical compositions, which comprise anamount of the ATR inhibitor effective to treat or prevent the diseasesor conditions described herein and a pharmaceutically acceptablecarrier, are another embodiment of the present invention.

The exact amount of compound required for treatment will vary fromsubject to subject, depending on the species, age, and general conditionof the subject, the severity of the infection, the particular agent, itsmode of administration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

In some embodiments, these compositions optionally further comprise oneor more additional therapeutic agents. For example, chemotherapeuticagents or other anti-proliferative agents may be combined with thecompounds of this invention to treat proliferative diseases and cancer.Examples of known agents with which these compositions can be combinedare listed above under the “Combination Therapies” section and alsothroughout the specification. Some embodiments provide a simultaneous,separate or sequential use of a combined preparation.

Modes of Administration and Dosage Forms

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in microencapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, eardrops, and eye drops are also contemplated asbeing within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

The compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes, but is not limited to, subcutaneous,intravenous, intramuscular, intra-articular, intra-synovial,intrasternal, intrathecal, intrahepatic, intralesional and intracranialinjection or infusion techniques. Preferably, the compositions areadministered orally, intraperitoneally or intravenously.

Sterile injectable forms of the compositions of this invention may beaqueous or oleaginous suspension. These suspensions may be formulatedaccording to techniques known in the art using suitable dispersing orwetting agents and suspending agents. The sterile injectable preparationmay also be a sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically-acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include, but arenot limited to, lactose and corn starch. Lubricating agents, such asmagnesium stearate, are also typically added. For oral administration ina capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions are required for oral use, the activeingredient is combined with emulsifying and suspending agents. Ifdesired, certain sweetening, flavoring or coloring agents may also beadded.

Alternatively, the pharmaceutical compositions of this invention may beadministered in the form of suppositories for rectal administration.These can be prepared by mixing the agent with a suitable non-irritatingexcipient that is solid at room temperature but liquid at rectaltemperature and therefore will melt in the rectum to release the drug.Such materials include, but are not limited to, cocoa butter, beeswaxand polyethylene glycols.

The pharmaceutical compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, the pharmaceutical compositions may beformulated in a suitable ointment containing the active componentsuspended or dissolved in one or more carriers. Carriers for topicaladministration of the compounds of this invention include, but are notlimited to, mineral oil, liquid petrolatum, white petrolatum, propyleneglycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax andwater. Alternatively, the pharmaceutical compositions can be formulatedin a suitable lotion or cream containing the active components suspendedor dissolved in one or more pharmaceutically acceptable carriers.Suitable carriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

For ophthalmic use, the pharmaceutical compositions may be formulated asmicronized suspensions in isotonic, pH adjusted sterile saline, or,preferably, as solutions in isotonic, pH adjusted sterile saline, eitherwith or without a preservative such as benzylalkonium chloride.Alternatively, for ophthalmic uses, the pharmaceutical compositions maybe formulated in an ointment such as petrolatum.

The pharmaceutical compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

The amount of protein kinase inhibitor that may be combined with thecarrier materials to produce a single dosage form will vary dependingupon the host treated, the particular mode of administration.Preferably, the compositions should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of the inhibitor can beadministered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of inhibitor will also depend upon the particular compound in thecomposition.

Administering with Another Agent

Depending upon the particular protein kinase-mediated conditions to betreated or prevented, additional drugs, which are normally administeredto treat or prevent that condition, may be administered together withthe compounds of this invention.

Those additional agents may be administered separately, as part of amultiple dosage regimen, from the protein kinase inhibitor-containingcompound or composition. Alternatively, those agents may be part of asingle dosage form, mixed together with the protein kinase inhibitor ina single composition.

Another aspect of this invention is directed towards a method oftreating cancer in a subject in need thereof, comprising the sequentialor co-administration of a compound of this invention or apharmaceutically acceptable salt thereof, and an anti-cancer agent. Insome embodiments, said anti-cancer agent is selected from Platinatingagents, such as Cisplatin, Oxaliplatin, Carboplatin, Nedaplatin, orSatraplatin and other derivatives; Topo I inhibitors, such asCamptothecin, Topotecan, irinotecan/SN38, rubitecan and otherderivatives; Antimetabolites, such as Folic family (Methotrexate,Pemetrexed and relatives); Purine family (Thioguanine, Fludarabine,Cladribine, 6-Mercaptopurine and relatives); Pyrimidine family(Cytarabine, Gemcitabine, 5-Fluorouracil and relatives); Alkylatingagents, such as Nitrogen mustards (Cyclophosphamide, Melphalan,Chlorambucil, mechlorethamine, Ifosfamide, and relatives); nitrosoureas(e.g. Carmustine); Triazenes (Dacarbazine, temozolomide); Alkylsulphonates (e.g. Busulfan); Procarbazine and Aziridines; Antibiotics,such as Hydroxyurea; Anthracyclines (doxorubicin, daunorubicin,epirubicin and other derivatives); Anthracenediones (Mitoxantrone andrelatives); Streptomyces family (Bleomycin, Mitomycin C, actinomycin)and Ultraviolet light.

Biological Samples

As inhibitors of ATR kinase, the compounds and compositions of thisinvention are also useful in biological samples. One aspect of theinvention relates to inhibiting ATR kinase activity in a biologicalsample, which method comprises contacting said biological sample with acompound described herein or a composition comprising said compound. Theterm “biological sample”, as used herein, means an in vitro or an exvivo sample, including, without limitation, cell cultures or extractsthereof; biopsied material obtained from a mammal or extracts thereof;and blood, saliva, urine, feces, semen, tears, or other body fluids orextracts thereof. The term “compounds described herein” includescompounds of formula III.

Inhibition of ATR kinase activity in a biological sample is useful for avariety of purposes that are known to one of skill in the art. Examplesof such purposes include, but are not limited to, blood transfusion,organ-transplantation, and biological specimen storage.

Study of Protein Kinases

Another aspect of this invention relates to the study of protein kinasesin biological and pathological phenomena; the study of intracellularsignal transduction pathways mediated by such protein kinases; and thecomparative evaluation of new protein kinase inhibitors. Examples ofsuch uses include, but are not limited to, biological assays such asenzyme assays and cell-based assays.

The activity of the compounds as protein kinase inhibitors may beassayed in vitro, in vivo or in a cell line. In vitro assays includeassays that determine inhibition of either the kinase activity or ATPaseactivity of the activated kinase. Alternate in vitro assays quantitatethe ability of the inhibitor to bind to the protein kinase and may bemeasured either by radiolabelling the inhibitor prior to binding,isolating the inhibitor/kinase complex and determining the amount ofradiolabel bound, or by running a competition experiment where newinhibitors are incubated with the kinase bound to known radioligands.Detailed conditions for assaying a compound utilized in this inventionas an inhibitor of ATR is set forth in the Examples below.

Another aspect of the invention provides a method for modulating enzymeactivity by contacting a compound described herein with ATR kinase.

Methods of Treatment

In one aspect, the present invention provides a method for treating orlessening the severity of a disease, condition, or disorder where ATRkinase is implicated in the disease state. In another aspect, thepresent invention provides a method for treating or lessening theseverity of an ATR kinase disease, condition, or disorder whereinhibition of enzymatic activity is implicated in the treatment of thedisease. In another aspect, this invention provides a method fortreating or lessening the severity of a disease, condition, or disorderwith compounds that inhibit enzymatic activity by binding to the ATRkinase. Another aspect provides a method for treating or lessening theseverity of a kinase disease, condition, or disorder by inhibitingenzymatic activity of ATR kinase with an ATR kinase inhibitor.

One aspect of the invention relates to a method of inhibiting ATR kinaseactivity in a patient, which method comprises administering to thepatient a compound described herein, or a composition comprising saidcompound. In some embodiments, said method is used to treat or prevent acondition selected from proliferative and hyperproliferative diseases,such as cancer.

Another aspect of this invention provides a method for treating,preventing, or lessening the severity of proliferative orhyperproliferative diseases comprising administering an effective amountof a compound, or a pharmaceutically acceptable composition comprising acompound, to a subject in need thereof. In some embodiments, saidsubject is a patient. The term “patient”, as used herein, means ananimal, preferably a human.

In some embodiments, said method is used to treat or prevent cancer. Insome embodiments, said method is used to treat or prevent a type ofcancer with solid tumors. In yet another embodiment, said cancer isselected from the following cancers: Oral: buccal cavity, lip, tongue,mouth, pharynx; Cardiac: sarcoma (angiosarcoma, fibrosarcoma,rhabdomyosarcoma, liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma andteratoma; Lung: bronchogenic carcinoma (squamous cell or epidermoid,undifferentiated small cell, undifferentiated large cell,adenocarcinoma), alveolar (bronchiolar) carcinoma, bronchial adenoma,sarcoma, lymphoma, chondromatous hamartoma, mesothelioma;Gastrointestinal: esophagus (squamous cell carcinoma, larynx,adenocarcinoma, leiomyosarcoma, lymphoma), stomach (carcinoma, lymphoma,leiomyosarcoma), pancreas (ductal adenocarcinoma, insulinoma,glucagonoma, gastrinoma, carcinoid tumors, vipoma), small bowel or smallintestines (adenocarcinoma, lymphoma, carcinoid tumors, Karposi'ssarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma), largebowel or large intestines (adenocarcinoma, tubular adenoma, villousadenoma, hamartoma, leiomyoma), colon, colon-rectum, colorectal; rectum,Genitourinary tract: kidney (adenocarcinoma, Wilm's tumor[nephroblastoma], lymphoma), bladder and urethra (squamous cellcarcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Skin:malignant melanoma, basal cell carcinoma, squamous cell carcinoma,Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma,angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillarythyroid carcinoma, follicular thyroid carcinoma; undifferentiatedthyroid cancer, medullary thyroid carcinoma, multiple endocrineneoplasia type 2A, multiple endocrine neoplasia type 2B, familialmedullary thyroid cancer, pheochromocytoma, paraganglioma; and Adrenalglands: neuroblastoma.

In some embodiments, the cancer is selected from the cancers describedherein. In some embodiments, said cancer is lung cancer, head and neckcancer, pancreatic cancer, gastric cancer, or brain cancer.

In certain embodiments, an “effective amount” of the compound orpharmaceutically acceptable composition is that amount effective inorder to treat said disease. The compounds and compositions, accordingto the method of the present invention, may be administered using anyamount and any route of administration effective for treating orlessening the severity of said disease.

One aspect provides a method for inhibiting ATR in a patient comprisingadministering a compound described herein as described herein. Anotherembodiment provides a method of treating cancer comprising administeringto a patient a compound described herein, wherein the variables are asdefined herein.

Some embodiments comprising administering to said patient an additionaltherapeutic agent selected from a DNA-damaging agent; wherein saidadditional therapeutic agent is appropriate for the disease beingtreated; and said additional therapeutic agent is administered togetherwith said compound as a single dosage form or separately from saidcompound as part of a multiple dosage form.

In some embodiments, said DNA-damaging agent is selected from ionizingradiation, radiomimetic neocarzinostatin, a platinating agent, a Topo Iinhibitor, a Topo II inhibitor, an antimetabolite, an alkylating agent,an alkyl sulphonates, an antimetabolite, or an antibiotic. In otherembodiments, said DNA-damaging agent is selected from ionizingradiation, a platinating agent, a Topo I inhibitor, a Topo II inhibitor,or an antibiotic.

Examples of Platinating agents include Cisplatin, Oxaliplatin,Carboplatin, Nedaplatin, Satraplatin and other derivatives. Otherplatinating agents include Lobaplatin, and Triplatin. Other platinatingagents include Tetranitrate, Picoplatin, Satraplatin, ProLindac andAroplatin.

Examples of Topo I inhibitor include Camptothecin, Topotecan,irinotecan/SN38, rubitecan and other derivatives. Other Topo Iinhibitors include Belotecan.

Examples of Topo II inhibitors include Etoposide, Daunorubicin,Doxorubicin, Aclarubicin, Epirubicin, Idarubicin, Amrubicin,Pirarubicin, Valrubicin, Zorubicin and Teniposide.

Examples of Antimetabolites include members of the Folic family, Purinefamily (purine antagonists), or Pyrimidine family (pyrimidineantagonists). Examples of the Folic family include methotrexate,pemetrexed and relatives; examples of the Purine family includeThioguanine, Fludarabine, Cladribine, 6-Mercaptopurine, and relatives;examples of the Pyrimidine family include Cytarabine, gemcitabine,5-Fluorouracil (5FU) and relatives.

Some other specific examples of antimetabolites include Aminopterin,Methotrexate, Pemetrexed, Raltitrexed, Pentostatin, Cladribine,Clofarabine, Fludarabine, Thioguanine, Mercaptopurine, Fluorouracil,Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine, Gemcitabine,Azacitidine and Hydroxyurea.

Examples of alkylating agents include Nitrogen mustards, Triazenes,alkyl sulphonates, Procarbazine and Aziridines. Examples of Nitrogenmustards include Cyclophosphamide, Melphalan, Chlorambucil andrelatives; examples of nitrosoureas include Carmustine; examples oftriazenes include Dacarbazine and temozolomide; examples of alkylsulphonates include Busulfan.

Other specific examples of alkylating agents include Mechlorethamine,Cyclophosphamide, Ifosfamide; Trofosfamide, Chlorambucil, Melphalan,Prednimustine, Bendamustine, Uramustine, Estramustine, Carmustine,Lomustine, Semustine, Fotemustine, Nimustine, Ranimustine, Streptozocin,Busulfan, Mannosulfan, Treosulfan, Carboquone, ThioTEPA, Triaziquone,Triethylenemelamine, Procarbazine, Dacarbazine, Temozolomide,Altretamine, Mitobronitol, Actinomycin, Bleomycin, Mitomycin andPlicamycin.

Examples of antibiotics include Mitomycin, Hydroxyurea; Anthracyclines,Anthracenediones, Streptomyces family. Examples of Anthracyclinesinclude doxorubicin, daunorubicin, epirubicin and other derivatives;examples of Anthracenediones include Mitoxantrone and relatives;examples of Streptomyces family include Bleomycin, Mitomycin C, andactinomycin.

In certain embodiments, said platinating agent is Cisplatin orOxaliplatin; said Topo I inhibitor is Camptothecin; said Topo IIinhibitor is Etoposide; and said antibiotic is Mitomycin. In otherembodiments, said platinating agent is selected from Cisplatin,Oxaliplatin, Carboplatin, Nedaplatin, or Satraplatin; said Topo Iinhibitor is selected from Camptothecin, Topotecan, irinotecan/SN38,rubitecan; said Topo II inhibitor is selected from Etoposide; saidantimetabolite is selected from a member of the Folic Family, the PurineFamily, or the Pyrimidine Family; said alkylating agent is selected fromnitrogen mustards, nitrosoureas, triazenes, alkyl sulfonates,Procarbazine, or aziridines; and said antibiotic is selected fromHydroxyurea, Anthracyclines, Anthracenediones, or Streptomyces family.

Another embodiment provides a method of promoting cell death in cancercells comprising administering to a patient a compound described herein,or a composition comprising said compound.

Yet another embodiment provides a method of preventing cell repair ofDNA damage in cancer cells comprising administering to a patient acompound described herein, or a composition comprising said compound.Yet another embodiment provides a method of preventing cell repaircaused by of DNA damage in cancer cells comprising administering to apatient a compound of formula III, or composition comprising saidcompound.

Another embodiment provides a method of sensitizing cells to DNAdamaging agents comprising administering to a patient a compounddescribed herein, or a composition comprising said compound.

In some embodiments, the method is used on a cancer cell having defectsin the ATM signaling cascade. In some embodiments, said defect isaltered expression or activity of one or more of the following: ATM,p53, CHK2, MRE11, RAD50, NBS1, 53BP1, MDC1 or H2AX. In anotherembodiment, the cell is a cancer cell expressing DNA damaging oncogenes.In some embodiments, said cancer cell has altered expression or activityof one or more of the following: K-Ras, N-Ras, H-Ras, Raf, Myc, Mos,E2F, Cdc25A, CDC4, CDK2, Cyclin E, Cyclin A and Rb.

Yet another embodiment provides use of a compound described herein as aradio-sensitizer or a chemo-sensitizer.

Yet other embodiment provides use of a compound of formula III as asingle agent (monotherapy) for treating cancer. In some embodiments, thecompounds of formula III are used for treating patients having cancerwith a DNA-damage response (DDR) defect. In other embodiments, saiddefect is a mutation or loss of ATM, p53, CHK2, MRE11, RAD50, NBS1,53BP1, MDC1, or H2AX.

Schemes

The compounds of the disclosure may be prepared in light of thespecification using steps generally known to those of ordinary skill inthe art. Those compounds may be analyzed by known methods, including butnot limited to LCMS (liquid chromatography mass spectrometry) and NMR(nuclear magnetic resonance). Below are a set of generic schemes thatillustrate generally how to prepare the compounds of the presentdisclosure.

Scheme A depicts a general method for making compounds of Formula III.The carboxylic acid of pyridine A is transformed into either an amide ora functional group deriving from the carboxylic acid, using standardconditions to give compounds of Formula A-i. In certain cases compoundsof Formula A-i can be functionalised directly via either an SNArreaction or a range of known metal mediated reactions such as, but notlimited to, Suzuki reactions, Sonagashira reactions, Stille couplings,Buchwald-Hartwig reactions and carbonylation reactions to give compoundsof Formula III. Alternatively, compounds of Formula A-i are protectedwith a suitable amine protecting group PG such as, but not limited toBOC (^(t)Butyl Carbamate), to give compounds of Formula A-ii. J¹substituents on R¹ can undergo further functionalisation by reactionsknown to those skilled in the art such as, but not limited to,bromination reactions and nucleophilic displacement reactions. Compoundsof Formula A-ii can be functionalised via either an SNAr reaction or arange of known metal mediated reactions such as, but not limited to,Suzuki reactions, Sonagashira reactions, Stille couplings,Buchwald-Hartwig reactions and carbonylation reactions to give compoundsof Formula A-iii. J^(Q) or J^(Q1) substituents on R² can undergo furtherfunctionalisation by reactions known to those skilled in the art suchas, but not limited to, acylation reactions and amide bond formationreactions. Removal of the nitrogen protecting group PG′ from compoundsof Formula A-iii under standard conditions known to those skilled in theart such as, but not limited to, treatment with HCl or TFA gives rise tocompounds of Formula III. In addition, J^(Q) or J^(Q1) substituents onR² or J¹ substituents on R¹ can undergo further functionalisation byreactions known to those skilled in the art such as, but not limited to,bromination reactions, nucleophilic displacement reactions acylationreactions and amide bond formation reactions at this stage.

Scheme B depicts a general method for making compounds of Formula IIIwhere (L)_(n)R¹ can derive from an alkyne functional group.Iodo-pyridine B is reacted with a suitably protected alkyne underSonagashira conditions and gives rise to compounds of the Formula B-i.Suitable alkyne protecting groups PG′ include, but are not limited to,TMS, TES or TIPS. Compounds of Formula B-i are then protected with asuitable amine protecting group PG such as, but not limited to BOC(^(t)Butyl Carbamate), to give compounds of Formula B-ii. Compounds ofthis example are then selectively deprotected under standard conditionsknown to those skilled in the art such as, but not limited to, treatmentwith aqueous base to remove the alkyne protecting group PG′ yieldingcompounds of Formula B-iii. These compounds are further elaborated underreaction conditions known in the art to give compounds of Formula A-ii.Any J¹ substituents on R¹ can undergo further functionalisation byreactions such as, but not limited to, bromination reactions andnucleophilic displacement reactions. Compounds of Formula A-ii can befunctionalised via either an SNAr reaction or a range of known metalmediated reactions such as, but not limited to, Suzuki reactions,Sonagashira reactions, Stille couplings, Buchwald-Hartwig reactions andcarbonylation reactions to give compounds of Formula A-iii. J^(Q) orJ^(Q1) substituents on R² can undergo further functionalisation byreactions such as, but not limited to, acylation reactions and amidebond formation reactions. Removal of the nitrogen protecting group PG′from compounds of Formula A-iii under standard conditions gives rise tocompounds of Formula III. In addition, J^(Q) or J^(Q1) substituents onR² or J¹ substituents on R¹ can undergo further functionalisation byreactions known to those skilled in the art such as, but not limited to,bromination reactions, nucleophilic displacement reactions acylationreactions and amide bond formation reactions at this stage.

Scheme C depicts a general method for making compounds of Formula III.Compounds of the Formula A-ii can be prepared by using the methodologiesoutlined in Schemes A and B above. These compounds are reacted understandard conditions known to those skilled in the art such as but notlimited to, Palladium catalysed boronate formation to give compounds ofFormula C-i. These compounds can undergo metal mediated couplingreactions such as, but not limited to, the Suzuki reaction to givecompounds of the Formula A-iii. J^(Q) or J^(Q1) substituents on R² canundergo further functionalisation by reactions known to those skilled inthe art such as, but not limited to, acylation reactions and amide bondformation reactions. Removal of the nitrogen protecting group PG′ fromcompounds of Formula A-iii under standard conditions known to thoseskilled in the art such as, but not limited to, treatment with HCl orTFA gives rise to compounds of Formula III. In addition, J^(Q) or J^(Q1)substituents on R² or R¹ substituents on R¹ can undergo furtherfunctionalisation by reactions known to those skilled in the art suchas, but not limited to, bromination reactions, nucleophilic displacementreactions acylation reactions and amide bond formation reactions at thisstage.

EXAMPLES

In order that this invention be more fully understood, the followingpreparative and testing examples are set forth. These examples are forthe purpose of illustration only and are not to be construed as limitingthe scope of the invention in any way. ¹H-NMR spectra were recorded at400 MHz using a Bruker DPX 400 instrument. Mass spec. samples wereanalyzed on a MicroMass Quattro Micro mass spectrometer operated insingle MS mode with electrospray ionization.

Example 1 2-Amino-N-phenyl-5-(3-pyridyl)pyridine-3-carboxamide (CompoundIII-7)

Method A Step 1: 2-Amino-5-bromo-N-phenylpyridine-3-carboxamide

A mixture of 2-amino-5-bromo-pyridine-3-carboxylic acid (203 mg, 0.9354mmol), aniline (87.11 mg, 85.23 0.9354 mmol),1-methylsulfonylbenzotriazole (276.7 mg, 1.403 mmol) and DIPEA (241.8mg, 325.9 1.871 mmol) in THF (2.0 mL) was heated in the microwave at160° C. for 10 mins. The reaction mixture was concentrated and theresidue partitioned between EtOAc and 2M NaOH. The organic phase waswashed with brine, dried (MgSO₄) and concentrated in vacuo to give theproduct as a yellow solid (174 mg, 64% Yield). ¹H NMR (400.0 MHz, DMSO)δ 7.77 (br s, 2H), 8.18 (d, 1H) and 8.32 (d, 1H); ppm; MS (ES⁺) 293.94.

Step 2: 2-Amino-N-phenyl-5-(3-pyridyl)pyridine-3-carboxamide

A mixture of 2-amino-5-bromo-N-phenyl-pyridine-3-carboxamide (230 mg,0.5511 mmol), 3-pyridylboronic acid (74.45 mg, 0.6057 mmol),tetrakis(triphenylphosphine) palladium(0) (31.85 mg, 0.02756 mmol) andpotassium carbonate (551.5 μL of 2 M, 1.103 mmol) in dioxane (1.40 mL)was heated in the microwave at 170° C. for 10 minutes. The reaction wasfiltered to remove Pd residues and partitioned between EtOAc and water.The organic phase was dried (Na₂SO₄), filtered and concentrated andtriturated with MeCN. The resultant solid was crystallised from EtOAc togive the pure product as a pale brown solid (98 mg, 43% Yield). ¹H NMR(400.0 MHz, DMSO) δ 7.12 (1H, t), 7.23 (2H, brs), 7.38 (2H, t), 7.48(1H, dd), 7.69 (1H, s), 7.72 (1H, s), 8.14 (1H, dt), 8.42 (1H, d), 8.54(1H, dd), 8.56 (1H, d), 8.98 (1H, d), 10.28 (1H, s) ppm; MS (ES⁺) 290.7.

Example 25-(4-(Methylsulfonyl)phenyl)-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine(Compound III-3)

Method B Step 1:2-Amino-5-bromo-N′-(phenylcarbonyl)pyridine-3-carbohydrazide

TBTU (2.219 g, 6.912 mmol) and triethylamine (466.3 mg, 642.3 μL, 4.608mmol) were added to a solution of 2-amino-5-bromo-pyridine-3-carboxylicacid (1 g, 4.608 mmol) and benzohydrazide (752.9 mg, 5.530 mmol) in DMF(40.00 mL) and the resulting solution stirred at ambient temperatureover the weekend. The reaction was treated with water/EtOAc and theorganics separated. The organic layer was washed with water (3×), dried(MgSO₄), filtered and concentrated in vacuo to give a yellow oil whichwas triturated with ether and dried under high vacuum to give theproduct as an off-white solid that was used directly in the next stepwithout further purification (1.177 g, 62% Yield). MS (ES⁺) 336.86.

Step 2: 5-Bromo-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine

Polyphosphonic acid (4.006 mL of 84% w/v, 41.04 mmol) was heated to 100°C. and treated with 2-amino-N′-benzoyl-5-bromonicotinohydrazide (673 mg,1.626 mmol) in portions over a period of 25 minutes. The reactionmixture was allowed to stir for 2 hours at 110° C. and then allowed tocool to ambient temperature. Water was added and the mixture basified(using 1M NaOH) and extracted with DCM (×3). The organic layer wasseparated, washed with brine, dried (MgSO₄), filtered and concentratedin vacuo to give the product as a white solid (372 mg, 72% Yield). ¹HNMR (400.0 MHz, DMSO) δ 7.54 (s, 2H), 7.62-7.68 (m, 3H), 8.22-8.25 (m,2H), 8.32 (d, J=2.5 Hz, 1H) and 8.50 (d, J=2.5 Hz, 1H) ppm; MS (ES⁺)318.86.

Step 3: tert-ButylN-[5-bromo-3-(5-phenyl-1,3,4-oxadiazol-2-yl)-2-pyridyl]-N-tert-butoxycarbonyl-carbamate

5-bromo-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine (556 mg, 1.753mmol) was suspended in a mixture of DCM (8.31 mL) and THF (8.31 mL) andtreated with DMAP (21.42 mg, 0.1753 mmol) followed by portionwiseaddition of BOC anhydride (1.148 g, 1.208 mL, 5.259 mmol). The reactionwas allowed to stir at ambient temperature for 48 hours and thenconcentrated in vacuo, redissolved in DCM and washed with saturatedsodium bicarbonate, followed by brine. The organics were separated,dried (Na₂SO₄), filtered and concentrated in vacuo. The resultantresidue was purified by column chromatography (ISCO Companion™, 12 gcolumn, 0-30% EtOAc/Petroleum ether) to give the desired product (762mg, 84% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.27 (18H, s), 7.69 (3H, m),8.16 (2H, m), 8.95 (2H, m) ppm; MS (ES⁺) 520.01.

Step 4:5-(4-(Methylsulfonyl)phenyl)-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine

A mixture of tert-butylN-[5-bromo-3-(5-phenyl-1,3,4-oxadiazol-2-yl)-2-pyridyl]-N-tert-butoxycarbonyl-carbamate(250 mg, 0.4832 mmol), (4-methylsulfonylphenyl)boronic acid (116.0 mg,0.5798 mmol), 4-ditert-butylphosphanyl-N,N-dimethyl-aniline;dichloropalladium (3.421 mg, 0.004832 mmol) and K₂CO₃ (133.6 mg, 0.9664mmol) in Toluene (2.250 mL) and water (250.0 μL) was heated to 100° C.for 48 hours. The mixture was allowed to cool to ambient temperature anddiluted with DCM and water. The organic layer was separated, washed with1M NaOH solution, dried (Na₂SO₄), filtered and concentrated in vacuo.The resultant residue was triturated with DCM to give the desiredproduct as a peach solid (70 mg, 37% Yield). ¹H NMR (400.0 MHz, DMSO) δ3.27 (s, 3H), 7.64-7.69 (m, 5H), 8.01 (d, J=8.5 Hz, 2H), 8.07 (d, J=8.6Hz, 2H), 8.25-8.27 (m, 2H), 8.66 (d, J=2.5 Hz, 1H) and 8.72 (d, J=2.5Hz, 1H) ppm; MS (ES⁺) 392.9.

Example 35-(1-Ethylsulfonyl-3,6-dihydro-2H-pyridin-4-yl)-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine(Compound III-4)

Method C Step 1: tert-Butyl4-(6-(tert-butoxycarbonylamino)-5-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-3-yl)-5,6-dihydropyridine-1(2H)-carboxylate

A mixture of tert-butylN-[5-bromo-3-(5-phenyl-1,3,4-oxadiazol-2-yl)-2-pyridyl]-N-tert-butoxycarbonyl-carbamate(300 mg, 0.5799 mmol), tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate(215.2 mg, 0.6959 mmol), dichloropalladium; triethylphosphane (12.00 mg,0.02900 mmol) and Na₂CO₃ (184.4 mg, 1.740 mmol) in DMF (3.000 mL) washeated to 60° C. for 16 hours. Analysis indicated that there wasstarting material remaining so the reaction was heated to 140° C. in themicrowave for a further 2 hours. The reaction mixture was allowed tocool to ambient temperature, diluted with DCM and water and the twolayers separated. The organics were washed with 1M NaOH solution, dried(Na₂SO₄), filtered and concentrated in vacuo. The resultant residue waspurified by column chromatography (EtOAc/Petroleum ether) to give thedesired product that was used directly in the next step without furtherpurification (142 mg, 47% Yield). MS (ES⁺) 302.31.

Step 2:5-(1-Ethylsulfonyl-3,6-dihydro-2H-pyridin-4-yl)-3-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-2-amine

tert-Butyl4-[6-(tert-butoxycarbonylamino)-5-(5-phenyl-1,3,4-oxadiazol-2-yl)-3-pyridyl]-3,6-dihydro-2H-pyridine-1-carboxylate(141.7 mg, 0.2727 mmol) was dissolved in DCM (2 mL) and treated with TFA(373.1 mg, 252.1 μL, 3.272 mmol) at ambient temperature. The reactionwas allowed to stir at ambient temperature for 3 hours and thenconcentrated in vacuo. The resultant residue was redissolved in DCM andtreated with triethylamine (55.19 mg, 76.02 μL, 0.5454 mmol) followed byethanesulfonyl chloride (17.54 mg, 12.93 μL, 0.1364 mmol). The mixturewas allowed to stir at ambient temperature for 16 hours and thenconcentrated in vacuo. The resultant residue was purified by reversephase preparative HPLC [Waters Sunfire C18, 10 mM, 100 Å column,gradient 10%-95% B (solvent A: 0.05% TFA in water; solvent B: CH₃CN)over 16 minutes at 25 mL/min]. The fractions were collected, passedthrough a sodium bicarbonate cartridge and freeze-dried to give thetitle compound as an off-white solid (47 mg, 42% Yield). ¹H NMR (400.0MHz, DMSO) δ 1.25 (t, 3H), 2.67 (br s, 2H), 3.16 (br s, 2H), 3.47 (br s,2H), 3.94 (br s, 2H), 6.25 (br s, 1H), 7.44 (s, 2H), 7.64-7.69 (m, 3H),8.22-8.24 (m, 2H), 8.29 (s, 1H) and 8.39 (s, 1H) ppm; MS (ES⁺) 411.95.

Example 4[4-[6-Amino-5-(5-phenyl-1,3,4-oxadiazol-2-yl)-3-pyridyl]phenyl]-(1,4-diazepan-1-yl)methanone(Compound III-2)

Method D Step 1:4-(6-Amino-5-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-3-yl)benzoic acid

A mixture of tert-butylN-[5-bromo-3-(5-phenyl-1,3,4-oxadiazol-2-yl)-2-pyridyl]-N-tert-butoxycarbonyl-carbamate(198.7 mg, 0.3841 mmol), 4-boronobenzoic acid (63.74 mg, 0.3841 mmol)and Na₂CO₃ (81.42 mg, 0.7682 mmol) was suspended in MeCN (3.213mL)/water (3.213 mL) and degassed (nitrogen/vacuum cycles). The reactionwas then treated with tetrakis(triphenylphosphine)palladium(0) (44.39mg, 0.03841 mmol), degassed again and heated at 110° C. in the microwavefor 30 minutes. The reaction was treated with water (0.5 mL) and heatedin the microwave at 110° C. for a further 30 minutes. The reactionmixture was washed with DCM, the aqueous phase acidified to pH1 (byaddition of 1M HCl), and resulting precipitate filtered off and washedwith water to give the product as an orange solid (75 mg, 55% Yield). MS(ES⁺) 359.14.

Step 2:[4-[6-Amino-5-(5-phenyl-1,3,4-oxadiazol-2-yl)-3-pyridyl]phenyl]-(1,4-diazepan-1-yl)methanone

A solution of4-(6-amino-5-(5-phenyl-1,3,4-oxadiazol-2-yl)pyridin-3-yl)benzoic acid(75.4 mg, 0.2104 mmol), carbonyl diimidazole (68.23 mg, 0.4208 mmol),DMAP (2.570 mg, 0.02104 mmol) and DIPEA (81.58 mg, 109.9 μL 0.6312 mmol)in DMSO (1.282 mL) was treated with 1,4-diazepane (63.22 mg, 0.6312mmol) and the resulting solution stirred at ambient temperature for 16hours. The reaction was treated with TBTU (67.56 mg, 0.2104 mmol)followed by 1,4-diazepane (63.22 mg, 0.6312 mmol) and DMF (1.282 mL) andallowed to stir at 60° C. for a further 48 hours. The reaction wastreated with further TBTU (135.12 mg, (0.4208 mmol) and heated at 60° C.for a further 5 hours. The reaction mixture was diluted with EtOAc/waterand the two layers separated. The organic layer was separated, dried(MgSO₄), filtered and concentrated in vacuo. The resultant residue waspurified by reverse phase preparative HPLC [Waters Sunfire C18, 10 mM,100 Å column, gradient 10%-95% B (solvent A: 0.05% TFA in water; solventB: CH₃CN) over 16 minutes at 25 mL/min]. The fractions were collected,passed through a sodium bicarbonate cartridge and freeze-dried to givethe title compound (31 mg, 33% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.69(s, 1H), 1.79 (s, 1H), 2.68 (s, 1H), 2.79-2.86 (m, 2H), 2.98 (t, J=4.9Hz, 1H), 3.42 (dd, J=5.9, 12.8 Hz, 2H), 3.67 (d, J=5.3 Hz, 2H), 7.50(dd, J=3.4, 7.9 Hz, 2H), 7.56 (s, 2H), 7.63-7.71 (m, 3H), 7.84 (q, J=3.9Hz, 2H), 8.25-8.27 (m, 3H), 8.59 (d, J=2.3 Hz, 1H) and 8.66 (d, J=2.2Hz, 1H) ppm; MS (ES⁺) 441.2.

Example 55-(4-Isopropylsulfonylphenyl)-3-[5-[4-(methylaminomethyl)phenyl]-1,3,4-oxadiazol-2-yl]pyridin-2-amine(Compound III-11)

Method E Step 1: tert-ButylN-[[4-[[(2-amino-5-bromo-pyridine-3-carbonyl)amino]carbamoyl]phenyl]methyl]-N-methyl-carbamate

2-amino-5-bromo-pyridine-3-carboxylic acid (776.9 mg, 3.580 mmol) andtriethylamine (724.5 mg, 997.9 μL 7.160 mmol) were added to a stirredsolution of tert-butylN-[[4-(hydrazinecarbonyl)phenyl]methyl]-N-methyl-carbamate (1 g, 3.580mmol in DMF (8 mL). To the stirred slurry was added TBTU (1.724 g, 5.370mmol) and the mixture stirred at ambient for 60 hours. The mixture wasdiluted with EtOAc/water and the layers separated. The mixture wasextracted with ethyl acetate (×3) and the combined extracts washed withsaturated aqueous NaHCO₃ (×1) and brine (×1), dried (MgSO₄), filteredand concentrated in vacuo. The resultant residue was purified by columnchromatography (ISCO Companion™, 12 g column, 0-100% EtOAc/Petroleumether) and the product containing fractions concentrated in vacuo. Theresidue was triturated from acetonitrile to give the desired sub-titleproduct as a cream solid (992 mg, 58% Yield). ¹H NMR (400.0 MHz, DMSO) δ1.39-1.45 (m, 9H), 2.80 (s, 3H), 4.45 (s, 2H), 7.28 (s, 2H), 7.35 (d,J=8.2 Hz, 2H), 7.90 (d, J=7.8 Hz, 2H), 8.20 (d, J=2.4 Hz, 1H), 8.24 (d,J=2.2 Hz, 1H), 10.50 (s, 1H) and 10.54 (s, 1H) ppm; MS (ES⁺) 480.2.

Step 2: tert-ButylN-[[4-[5-(2-amino-5-bromo-3-pyridyl)-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate

tert-ButylN-[[4-[[(2-amino-5-bromo-pyridine-3-carbonyl)amino]carbamoyl]phenyl]methyl]-N-methyl-carbamate(992.3 mg, 2.074 mmol) was dissolved in dry MeCN (15 mL) and cooled inan ice bath under a nitrogen atmosphere. DIPEA (804.2 mg, 1.084 mL,6.222 mmol) was added via syringe followed bydibromo(triphenyl)phosphorane (1.185 g, 2.696 mmol) portionwise and thereaction allowed to warm to ambient temperature over 15 hours. A furtherportion of MeCN (15 mL) and dibromo(triphenyl)phosphorane (437.7 mg,1.037 mmol) were added and the reaction mixture stirred at ambienttemperature for a 10 minutes. The precipitate was isolated by filtrationand the filtrate concentrated in vacuo. The filtrate was re-dissolved inMeCN (15 mL) and cooled to 0° C. DIPEA (804.2 mg, 1.084 mL, 6.222 mmol)was added followed by dibromo(triphenyl)phosphorane (437.7 mg, 1.037mmol) and the reaction mixture stirred at 0° C. for 30 minutes. Thereaction mixture was concentrated in vacuo and the resultant residuepurified by column chromatography (ISCO Companion™, 40 g column, 0-50%EtOAc/Petroleum ether) and the product containing fractions concentratedin vacuo. The residue was combined with the previously obtainedprecipitate to give the sub-title product as a white solid (682 mg, 63%Yield corrected for residual OPPh₃). ¹H NMR (400.0 MHz, DMSO) δ1.39-1.46 (d, 9H), 4.48 (d, J=10.5 Hz, 2H), 7.46 (d, J=8.1 Hz, 2H), 8.22(d, J=8.2 Hz, 2H), 8.32 (d, J=2.4 Hz, 1H) and 8.49 (d, J=2.4 Hz, 1H)ppm; MS (ES⁺) 462.1.

Step 3: tert-ButylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-bromo-3-pyridyl]-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate

tert-ButylN-[[4-[5-(2-amino-5-bromo-3-pyridyl)-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate(681.59 mg, 1.310 mmol) was suspended in DCM (9 mL) and THF (9 mL) andDMAP (16.00 mg, 0.1310 mmol) was added, BOC₂O (857.7 mg, 902.8 μL, 3.930mmol) was added in portions and the reaction allowed to stir at ambienttemperature for 6 hours. DIPEA (338.6 mg, 456.3 μL, 2.620 mmol) wasadded and reaction mixture stirred at ambient temperature for 65 hours.A further portion of BOC₂O (571.8 mg, 2.620 mmol) was added and thereaction was heated at 60° C. for 17 hours. The solvent was removed invacuo and the residue re-dissolved in DCM. The organic layer was washedwith saturated aqueous NaHCO₃ (×4) and brine (×1), dried (MgSO₄),filtered and concentrated in vacuo. The resultant residue was purifiedby column chromatography (ISCO Companion™, 24 g column, 0-25%EtOAc/Petroleum ether) to give the sub-title compound as a yellow foam(711 mg, 82% Yield). NMR (400.0 MHz, DMSO) δ 1H NMR (400.0 MHz, DMSO) d1.26 (s, 18H), 1.37-1.45 (m, 9H), 2.85 (br s, 3H), 4.49 (s, 2H), 7.50(d, 2H), 8.15 (d, J=8.1 Hz, 2H) and 8.95 (d, J=2.7 Hz, 2H) ppm; MS (ES)562.1 (m-BOC).

Step 4: tert-ButylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-(4-isopropylsulfonylphenyl)-3-pyridyl]-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate

tert-ButylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-bromo-3-pyridyl]-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate(120 mg, 0.1817 mmol) was dissolved in DMF (1.2 mL) and2-(4-isopropylsulfonylphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane(84.57 mg, 0.2726 mmol) and Pd(PPh₃)₂Cl₂ (7.516 mg, 0.01817 mmol) wereadded. 2M aqueous Na₂CO₃ (272.6 μL, 0.5451 mmol) was added and thereaction heated at 80° C. for 1 hour in a sealed tube. The reactionmixture was cooled to ambient temperature and diluted with EtOAc/water.The layers were separated and the aqueous layer extracted with EtOAc(×2). The combined organic extracts were washed with water (×3), brine(×2), dried (MgSO₄), filtered and concentrated in vacuo. The resultantresidue was purified by column chromatography (ISCO Companion™, 4 gcolumn, 0-50% EtOAc/Petroleum ether) to give the impure sub-titlecompound as a yellow oil (136 mg). This was used directly in the nextstep with no further purification.

Step 5:5-(4-Isopropylsulfonylphenyl)-3-[5-[4-(methylaminomethyl)phenyl]-1,3,4-oxadiazol-2-yl]pyridin-2-amine

TFA (1.011 g, 683.1 μL, 8.870 mmol) was added to a stirred solution oftert-butylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-(4-isopropylsulfonylphenyl)-3-pyridyl]-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate(135.5 mg, 0.1774 mmol) in DCM (5 mL) and the reaction allowed to stirat ambient temperature for 2 hours. The reaction mixture wasconcentrated in vacuo and triturated from diethyl ether. The resultantprecipitate was isolated by filtration and re-dissolved in DCM/MeOH. Thefiltrate was passed through a sodium bicarbonate cartridge. The filtratewas passed through a SCX2 column and washed with MeOH. The product waseluted with 3.5 M ammonia in MeOH and the fractions concentrated invacuo. The residue was titrated from DCM/diethyl ether to give the titlecompound as a cream solid (26 mg, 32% Yield). ¹H NMR (400.0 MHz, DMSO) δ1H NMR (400.0 MHz, DMSO) ¹H d 1.20 (d, J=6.8 Hz, 6H), 2.54 (s, 3H),3.48-3.50 (m, 1H), 4.13 (s, 2H), 7.70 (d, J=8.2 Hz, 3H), 7.93 (d, J=7.7Hz, 3H), 8.08 (d, J=8.5 Hz, 2H), 8.31 (d, J=8.3 Hz, 2H), 8.68 (d, J=2.5Hz, 1H) and 8.74 (d, J=2.5 Hz, 1H) ppm; MS (ES⁺) 464.3.

The following compounds can also be prepared using similar methodologyto that outlined in Method E:

3-[5-[4-(Methylaminomethyl)phenyl]-1,3,4-oxadiazol-2-yl]-5-(3-thienyl)pyridin-2-amine(Compound III-9)

¹H NMR (400.0 MHz, DMSO)S 2.62-2.68 (m, 3H), 4.28 (t, J=5.9 Hz, 2H),7.51 (s, 1H), 7.69-7.76 (m, 4H), 7.97 (t, J=2.1 Hz, 1H), 8.35 (d, J=8.3Hz, 2H), 8.59 (d, J=2.4 Hz, 1H), 8.70 (d, J=2.4 Hz, 1H) and 8.89 (s, 2H)ppm; MS (ES⁺) 364.2.

Example 65-(5-Isopropylsulfonyl-2-pyridyl)-3-[5-[4-(methylaminomethyl)phenyl]-1,3,4-oxadiazol-2-yl]pyridin-2-amine(Compound III-10)

Method F Step 1: tert-ButylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-pyridyl]-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate

A mixture of tert-butylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-bromo-3-pyridyl]-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate(100 mg, 0.1514 mmol), bis(pinacolato)diboron (57.67 mg, 0.2271 mmol)and KOAc (44.58 mg, 0.4542 mmol) in dioxane (2 mL) was degassed andplaced under an atmosphere of nitrogen. PdCl₂(dppf).DCM (12.36 mg,0.01514 mmol) added in one portion and the reaction mixture heated at95° C. for 1.5 hours. The reaction was cooled to ambient temperature andused directly in the next step.

Step 2: tert-ButylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-(5-isopropylsulfonyl-2-pyridyl)-3-pyridyl]-1,3,4-oxadiazol-2-yl]phenyl]methyl]-N-methyl-carbamate

The reaction mixture from Method F Step 1 was diluted with DMF (2 mL) an2-bromo-5-isopropylsulfonyl-pyridine [Compound I-7a; prepared accordingto Method F-1] (48 mg, 0.1817 mmol), Pd(PPh₃)₂Cl₂ (6.263 mg, 0.01514mmol) and 2M aqueous Na₂CO₃ (227.1 μL, 0.4542 mmol) were added. Thereaction was degassed with nitrogen and stirred at 95° C. for 2 hours ina sealed tube. The reaction mixture was partitioned between EtOAc andbrine and the aqueous layer extracted with EtOAc (×2). The combinedorganic extracts were washed with brine (×3), dried (MgSO₄), filteredand concentrated in vacuo. The crude residue was used in thedeprotection step without further purification.

Step 3:5-(5-Isopropylsulfonyl-2-pyridyl)-3-[5-[4-(methylaminomethyl)phenyl]-1,3,4-oxadiazol-2-yl]pyridin-2-amine

The residue from Method F Step 2 was dissolved in DCM (10 mL) and TFA (2mL, 25.96 mmol) was added. The reaction was stirred at ambienttemperature for 15 hours. The solvent was removed in vacuo and theresidue azeotroped with DCM (×2) and ether (×2). The material waspurified by reverse phase preparative HPLC [Waters Sunfire C18, 10 μM,100 Å column, gradient 10%-95% B (solvent A: 0.05% TFA in water; solventB: CH₃CN) over 16 minutes at 25 mL/min]. The fractions were collected,passed through a sodium bicarbonate cartridge and freeze-dried to givethe title compound as an off-white solid (11.4 mg, 16% Yield). ¹H NMR(400.0 MHz, DMSO) δ 1.23 (d, J=6.8 Hz, 6H), 2.30 (s, 3H), 3.56 (sept,1H), 3.77 (s, 2H), 7.60 (d, 2H), 7.91 (s, 2H), 8.16 (d, 2H), 8.29 (dd,1H), 8.36 (d, 1H), 9.00 (d, 1H), 9.03 (d, 1H) and 9.12 (d, 1H) ppm; MS(ES⁺) 465.3.

Example 6a 2-Bromo-5-isopropylsulfonyl-pyridine (Compound III-10a)

Method F-1 Step 1: 5-Isopropylsulfanylpyridin-2-ol

5-iodopyridin-2-ol (1 g, 4.525 mmol), propane-2-thiol (689.3 mg, 840.6μL, 9.050 mmol), ethylene glycol (842.9 mg, 757.3 μL, 13.58 mmol), CuI(172.4 mg, 0.9050 mmol) and potassium carbonate (1.877 g, 13.58 mmol) inIPA (7 mL) were heated under microwave conditions at 120° C. for 2hours. The mixture was filtered through celite washing with DCM and thefiltrate concentrated in vacuo. The residue was purified by columnchromatography (ISCO Companion™, 40 g column, eluting with 0 to 100%EtOAc/Petroleum Ether, loaded in DCM). The fractions were combined andconcentrated in vacuo and the residue redissolved in EtOAc (10 mL) andstirred with 0.5 g of 3-Mercaptopropyl ethyl sulfide Silica for 45minutes. The silica was removed by filtration and the solvent removed invacuo to give the sub-title compound as a beige oil which was usedwithout further purification.

Step 2: 2-Bromo-5-isopropylsulfanyl-pyridine

DMF (5 mL) was added slowly to a stirred mixture of5-isopropylsulfanylpyridin-2-ol (765.8 mg, 4.525 mmol) and POBr3 (5.189g, 18.10 mmol) at ambient temperature. After 10 minutes, the reactionmixture was warmed to 95° C. for 1 hour. The reaction mixture was cooledto ambient temperature and quenched by the slow addition of ice. Theaqueous layer was extracted with EtOAc (×3) and the combined organicswashed with saturated aqueous NaHCO₃ (×3), brine (×1), dried (MgSO₄),filtered and concentrated in vacuo. The residue was purified by columnchromatography (ISCO Companion™, 80 g column, eluting with 0 to 20%EtOAc/Petroleum Ether, loaded in DCM) to give the sub-title product asan off-white solid (661 mg, 63% Yield, 65% purity). ¹H NMR (400.0 MHz,DMSO) δ 1.24 (d, J=6.8 Hz, 6H), 3.58 (sept, 1H), 7.62 (d, J=8.0 Hz, 1H),7.78 (dd, J=2.6, 8.4 Hz, 1H) and 8.37 (d, J=2.5 Hz, 1H) ppm; MS (ES⁺)231.7.

Step 3: 2-Bromo-5-isopropylsulfonyl-pyridine

mCPBA (1.595 g, 7.118 mmol) was added in portions to a stirred solutionof 2-bromo-5-isopropylsulfanyl-pyridine (661 mg, 2.847 mmol) inanhydrous DCM (10 mL) at 0° C. under an atmosphere of nitrogen. Thereaction mixture was allowed to warm to ambient temperature over 1 hour.The reaction was quenched by the addition of 1M aqueous NaOH and thelayers separated. The aqueous phase was extracted with DCM (×3) and thecombined organic extracts washed with sodium thiosulfate (×2) and brine(×1). The combined organic extracts were dried (MgSO₄), filtered andconcentrated in vacuo.The residue was purified by column chromatography(ISCO Companion™, 40 g column, eluting with 0 to 100% EtOAc/Petrol,loaded in DCM) to give the sub-title product as a white solid (517 mg,69% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.19 (d, J=6.9 Hz, 6H), 3.58(sept, 1H), 7.99 (d, J=8.6 Hz, 1H), 8.18 (dd, J=2.6, 8.3 Hz, 1H) and8.82 (d, J=2.5 Hz, 1H) ppm; MS (ES⁺) 266.0.

Example 6b (3R)-3-(4-Bromophenyl)sulfonyltetrahydrofuran (CompoundIII-10b)

Method F-2 Step 1: (3R)-3-(4-Bromophenyl)sulfanyltetrahydrofuran

To a suspension of (3S)-3-iodotetrahydrofuran [prepared according to themethod outline in US2006004018] (1.2 g, 6.061 mmol) and K₂CO₃ (1.675 g,12.12 mmol) in DMF (12.00 mL) at ambient temperature was added4-bromobenzenethiol (1.146 g, 6.061 mmol). The resulting suspension wasstirred at this temperature overnight. Water was added and then theaqueous layer extracted with DCM (×3). The combined organic extractswere dried (MgSO₄), filtered and concentrated in vacuo to give thesub-title compound as a clear oil which was used directly in the nextstep without further purification.

Step 2: (3R)-3-(4-Bromophenyl)sulfonyltetrahydrofuran

The oil isolated in Method F-2 Step 1 was dissolved in DCM (12.00 mL)and 3-chlorobenzenecarboperoxoic acid (2.300 g, 13.33 mmol) was added.The resultant solution stirred at ambient temperature overnight.Saturated aqueous NaHCO₃ was added to the reaction mixture followed byextraction with DCM (3×50 mL). The combined organic extracts were washedwith brine (1×20 mL), (MgSO₄), filtered and concentrated in vacuo togive the sub-title compound as a white solid (1.0 g, 57% Yield). ¹H NMR(400.0 MHz, CDCl₃) δ 2.18-2.25 (m, 1H), 2.35-2.46 (m, 1H), 3.76-3.99 (m,4H), 4.19 (dd, 1H), 7.75-7.81 (m, 4H) ppm; MS (ES⁺) 293.0.

Example 6c 5-Isopropylsulfonylpyrazin-2-amine (Compound III-10c)

Method F-3 Step 1: 5-Isopropylsulfanylpyrazin-2-amine

Sodium-2-propanethiolate (2.256 g, 20.69 mmol) was added to a stirredsolution of 5-bromopyrazin-2-amine (3 g, 17.24 mmol) in DMF (15 mL) andheated at 100° C. under microwave conditions for 4 hours. A furtherportion of sodium-2-propanethiolate (2.256 g, 20.69 mmol) was added thereaction heated for a further 1 hour at 100° C. under microwaveconditions. Water was added and the mixture extracted with EtOAc (×2).The combined organic layers were washed with water (×3), brine (×1),dried, (MgSO₄), filtered and concentrated in vacuo to give the sub-titlecompound as a beige oil which was used without further purification.

Step 2: 5-Isopropylsulfonylpyrazin-2-amine

5-Isopropylsulfanylpyrazin-2-amine (3.5 g, 16.48 mmol) was dissolved indry DCM (65 mL) and the reaction mixture cooled in an ice bath.3-chlorobenzenecarboperoxoic acid (9.233 g, 41.20 mmol) was added inportions and the reaction mixture allowed to warm to ambient temperatureover 16 hours. The resultant precipitate was isolated by filtration andwashed with DCM. The filtrate was washed with 1M NaOH (×3) and saturatedsodium thiosulphate (×3). The organic extract was dried (MgSO₄),filtered and evaporated to dryness to give an orange solid which wastriturated from acetonitrile to give the sub-title compound as a whitesolid (650 mg, 19% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.18 (dd, J=7.0,9.5 Hz, 6H), 3.41-3.45 (m, 1H), 7.60 (s, 2H), 7.95-7.96 (m, 1H) and 8.38(d, J=1.1 Hz, 1H) ppm; MS (ES⁺) 202.2.

Step 3: 2-Bromo-5-isopropylsulfonyl-pyrazine

5-Isopropylsulfonylpyrazin-2-amine was added to a rapidly stirringmixture of hydrobromic acid in acetic acid (2.150 mL of 45% w/v, 11.96mmol) and acetic acid (649.6 μL) at 0° C. followed by the slow additionof bromine (1.433 g, 462.0 μL, 8.967 mmol) dropwise over a 10 minutes. Asolution of sodium nitrite (515.5 mg, 237.8 μL, 7.472 mmol) in water(2.275 mL) was added dropwise over 10 minutes and the reaction mixturewas stirred at 0° C. for a further 15 minutes. The reaction mixture wasbasified with 16 mL of saturated aqueous NaHCO₃ at 0° C. and extractedwith DCM (×3). The combined organic extracts were dried (MgSO₄),filtered and concentrated in vacuo. The residue was purified by columnchromatography (ISCO Companion™, 40 g column, eluting with 0 to 100%EtOAc/Petrol, loaded in DCM) to give the sub-title product as a creamsolid (349 mg, 44% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.24 (d, 6H), 3.69(sept, 1H). 9.04 (s, 1H) and 9.17 (s, 1H); MS (ES⁺) 267.0.

The following compounds can also be prepared using similar methodologyto that outlined in Method F:

3-[5-[4-(Methylaminomethyl)phenyl]-1,3,4-oxadiazol-2-yl]-5-[4-[(3R)-tetrahydrofuran-3-yl]sulfonylphenyl]pyridin-2-amine(Compound III-1)

¹H NMR (400.0 MHz, DMSO) δ 2.10-2.20 (m, 2H), 2.25 (s, 1H), 2.29 (s,2H), 3.60-3.90 (m, 5H), 4.00-4.10 (m, 1H), 4.20-4.30 (m, 1H), 7.45 (d,2H), 7.50 (br s, 2H), 7.99 (d, J=8.4 Hz, 2H), 8.08 (s, 2H), 8.20 (d,J=8.3 Hz, 2H), 8.70 (s, 1H) and 8.75 (s, 1H) ppm; MS (ES⁺) 492.1.

5-(5-Isopropylsulfonylpyrazin-2-yl)-3-[5-[4-(methylaminomethyl)phenyl]-1,3,4-oxadiazol-2-yl]pyridin-2-amine(Compound III-5)

¹H NMR (400.0 MHz, DMSO) δ 1.09 (d, J=6.8 Hz, 6H), 2.12 (s, 3H), 3.58(s, 1H), 3.60-3.68 (m, 1H), 3.70 (s, 2H), 7.51 (d, J=8.2 Hz, 2H), 7.99(d, J=8.2 Hz, 2H), 9.00 (m, 1H), 9.20 (m, 2H) and 9.51 (d, J=1.3 Hz, 1H)ppm; MS (ES⁺) 466.1.

Example 75-(5-Isopropylsulfonyl-2-pyridyl)-3-[3-[4-(methylaminomethyl)phenyl]isoxazol-5-yl]pyridin-2-amine(Compound III-12)

Method G Step 1: 5-Bromo-3-((trimethylsilyl)ethynyl)pyridin-2-amine

2-amino-5-bromo-3-iodopyridine (105.28 g, 0.3522 mol), CuI (0.6708 g,3.522 mmol) and PdCl₂(PPh₃)₂ (2.472 g, 3.522 mmol) were dissolved in dryTHF (210 mL) and degassed (vacuum/nitrogen cycles×4). Et₃N (107.0 g,0.1474 L, 1.057 mol) and TMS-acetylene (44.97 g, 0.06471 L, 0.4579 mol)were added and the resultant mixture was stirred at ambient temperaturefor 1¼ hours. The reaction was cooled in an ice-bath and ether (210 mL)was added. The resultant mixture was stirred at 0° C. for ½ hour,filtered through celite and the celite was washed copiously with ether.The filtrate was concentrated under reduced pressure to give abrown/black solid which was redissolved in the minimum necessary DCM andpurified by column chromatography (eluting with DCM). The productcontaining fractions were concentrated in vacuo and the residuerecrystallised from cyclohexane. The precipitate was isolated byfiltration to give the sub-title compound as a white powder (80.25 g,84% Yield). Analytical data identical to that of commercially availablecompound.

Step 2: tert-ButylN-[5-bromo-3-(2-trimethylsilylethynyl)-2-pyridyl]-N-tert-butoxycarbonyl-carbamate

BOC₂O (2.191 g, 2.306 mL, 10.04 mmol) and DMAP (40.88 mg, 0.3346 mmol)were added to a stirred solution of5-bromo-3-(2-trimethylsilylethynyl)pyridin-2-amine (900.8 mg, 3.346mmol) in anhydrous DCM (30 mL) at ambient temperature. The reaction wasallowed to stir at this temperature for 3 hours. The reaction mixturewas diluted with saturated aqueous NaHCO₃ and the layers separated. Theorganic extract was washed with NaHCO₃ (×1), brine (×1), dried (MgSO₄),filtered and concentrated in vacuo. The residue was purified by columnchromatography (ISCO Companion™, 40 g column, eluting with 0 to 10%EtOAc/Petroleum Ether, loaded in DCM) to give the sub-title product asan off-white solid (1.05 g, 67% Yield). ¹H NMR (400.0 MHz, DMSO) δ 0.00(s, 9H), 1.14 (s, 18H), 8.15 (d, J=2.4 Hz, 1H) and 8.44 (d, J=2.4 Hz,1H) ppm; MS (ES⁺) 471.0.

Step 3: tert-ButylN-(5-bromo-3-ethynyl-2-pyridyl)-N-tert-butoxycarbonyl-carbamate

2M aqueous Na₂CO₃ (639.0 μL, 1.278 mmol) was added to a stirred solutionof tert-butylN-[5-bromo-3-(2-trimethylsilylethynyl)-2-pyridyl]-N-tert-butoxycarbonyl-carbamate(500 mg, 1.065 mmol) in DMF (1.5 mL) and the resulting suspensionstirred at ambient temperature for 18 hours. The reaction mixture wasdiluted with EtOAc and water and the layers separated. The aqueous layerwas extracted with EtOAc (×2) and the combined organic extracts washedwith water (×3), brine (×2), dried (MgSO₄), filtered and concentrated invacuo to give the sub-title compound as an off-white solid (424 mg, 100%Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.34 (s, 18H), 4.78 (s, 1H), 8.38 (d,J=2.4 Hz, 1H) and 8.66 (d, J=2.4 Hz, 1H) ppm; MS (ES⁺) 399.1.

Step 4: tert-ButylN-[5-bromo-3-[3-(p-tolyl)isoxazol-5-yl]-2-pyridyl]-N-tert-butoxycarbonyl-carbamate

To a solution of tert-butylN-(5-bromo-3-ethynyl-2-pyridyl)-N-tert-butoxycarbonyl-carbamate (423 mg,1.065 mmol) and N-hydroxy-4-methyl-benzimidoyl chloride (212.5 mg, 1.065mmol) [Bioorganic & Medicinal Chemistry, 17(13), 4773-4785; 2009] in THF(10 mL) at ambient temperature was added Et₃N (129.3 mg, 178.1 μL, 1.278mmol) dropwise. The mixture was stirred at ambient temperature for 16hours then at 65° C. for 2 hours. The mixture was concentrated in vacuoand the residue redissolved in DCM and washed with saturated aqueousNaHCO₃ (×2) and brine (×1). The organic fraction was dried (MgSO₄),filtered and concentrated in vacuo. The residue was purified by columnchromatography (ISCO Companion™, 40 g column, eluting with 0 to 20%EtOAc/Petroleum Ether, loaded in DCM) to give the sub-title product as awhite solid (472 mg, 84% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.28 (s,18H), 2.39 (s, 3H), 7.39 (d, 2H), 7.60 (s, 1H), 7.68 (d, 2H), 8.73 (d,1H) and 8.82 (d, 1H) ppm; MS (ES⁺) 532.0.

Step 5: tert-ButylN-tert-butoxycarbonyl-N-[3-[3-(p-tolyl)isoxazol-5-yl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]carbamate

A mixture of tert-butylN-[5-bromo-3-[3-(p-tolyl)isoxazol-5-yl]-2-pyridyl]-N-tert-butoxycarbonyl-carbamate(472 mg, 0.8899 mmol), bis(pinacolato)diboron (339.0 mg, 1.335 mmol) andKOAc (262.0 mg, 2.670 mmol) in dioxane (10 mL) was degassed and placedunder an atmosphere of nitrogen. PdCl₂(dppf).DCM (72.67 mg, 0.08899mmol) added in one portion and the reaction mixture heated at 95° C. for1.5 hours. The reaction was cooled to ambient temperature and dilutedwith EtOAc/brine. The layers were separated and the aqueous layerextracted with EtOAc (×3). The combined organic extracts were dried(MgSO₄), filtered and concentrated in vacuo. The residue was passedthrough a pad of fluorosil eluting with 0-50% EtOAc/Petroleum Ether,loaded in DCM to give the sub-title product as an off-white solid (427mg, 83% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.26 (s, 18H), 1.37 (s, 12H),2.38 (s, 3H), 7.37 (d, J=8.1 Hz, 2H), 7.60 (s, 1H), 7.82 (d, J=8.1 Hz,2H), 8.51 (d, J=1.8 Hz, 1H) and 8.79 (d, J=1.6 Hz, 1H) ppm; MS (ES⁺)340.1.

Step 6: tert-ButylN-tert-butoxycarbonyl-N-[5-(5-isopropylsulfonyl-2-pyridyl)-3-[3-(p-tolyl)isoxazol-5-yl]-2-pyridyl]carbamate

A mixture of tert-butylN-tert-butoxycarbonyl-N-[3-[3-(p-tolyl)isoxazol-5-yl]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-2-pyridyl]carbamate(426 mg, 0.7377 mmol) and 2-bromo-5-isopropylsulfonyl-pyridine (233.8mg, 0.8852 mmol) in DMF (10 mL) was degassed and placed under anatmosphere of nitrogen. Pd(PPh₃)₂Cl₂ (30.51 mg, 0.07377 mmol) was addedand the reaction again degassed. 2M aqueous Na₂CO₃ (1.106 mL, 2.213mmol) was added and the reaction was stirred at 95° C. for 2 hours. Thereaction mixture was partitioned between EtOAc and brine and the aqueouslayer extracted with EtOAc (×3). The combined organic extracts werewashed with water (×3) and brine (×2), dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by column chromatography(ISCO Companion™, 40 g column, eluting with 0 to 50% EtOAc/PetroleumEther, loaded in DCM) to give the sub-title product as a yellow solid(344 mg, 74% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.24 (d, J=6.8 Hz, 6H),1.29 (s, 18H), 2.40 (s, 3H), 3.64 (sept, 1H), 7.40 (d, J=8.0 Hz, 2H),7.63 (s, 1H), 7.83 (d, J=8.1 Hz, 2H), 8.47 (dd, 1H), 8.58 (d, 1H), 9.12(d, J=2.2 Hz, 1H), 9.17 (d, 1H) and 9.42 (d, J=2.2 Hz, 1H) ppm; MS (ES⁺)635.2.

Step 7: tert-ButylN-[3-[3-[4-(bromomethyl)phenyl]isoxazol-5-yl]-5-(5-isopropylsulfonyl-2-pyridyl)-2-pyridyl]-N-tert-butoxycarbonyl-carbamate

tert-ButylN-tert-butoxycarbonyl-N-[5-(5-isopropylsulfonyl-2-pyridyl)-3-[3-(p-tolyl)isoxazol-5-yl]-2-pyridyl]carbamate(245 mg, 0.3860 mmol) was suspended in EtOAc (2.5 mL) followed by theaddition of NBS (89.31 mg, 0.5018 mmol) and AIBN (6.338 mg, 0.03860mmol). The mixture was heated to 85° C. and placed under a bright lampfor 2 hours. The reaction mixture was cooled to ambient temperature andwashed with saturated aqueous NaHCO₃/brine (1:1, ×2), dried (MgSO₄),filtered and concentrated in vacuo to give the impure sub-title compoundas a pale brown foam. This was used in the next step without any furtherpurification.

Step 8: tert-ButylN-[5-(5-isopropylsulfonyl-2-pyridyl)-3-[3-[4-(methylaminomethyl)phenyl]isoxazol-5-yl]-2-pyridyl]carbamate

tert-ButylN-[3-[3-[4-(bromomethyl)phenyl]isoxazol-5-yl]-5-(5-isopropylsulfonyl-2-pyridyl)-2-pyridyl]-N-tert-butoxycarbonyl-carbamate(275.5 mg, 0.3861 mmol) was taken into ethanol (1 mL)/DCM (1 mL) andthis solution added to stirred methylamine (3.634 g, 38.61 mmol) in ansealed tube. The reaction was stirred at ambient temperature for 2hours. The reaction mixture was diluted with DCM and washed withsaturated aqueous NaHCO₃/brine (1:1, ×2), dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by column chromatography(ISCO Companion™, 24 g column, eluting with 0 to 10% MeOH/DCM, loaded inDCM) to give the sub-title product as a beige solid (105.4 mg, 48%Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.23 (d, 6H), 1.32 (s, 9H), 2.30 (s,3H), 3.62 (sept, 1H), 3.72 (s, 2H), 7.46 (s, 1H), 7.50 (d, 2H), 7.87 (d,2H), 8.40 (dd, 1H), 8.49 (d, 1H), 8.91 (d, 1H), 9.11 (d, 1H), 9.28 (d,1H) and 10.06 (br s, 1H) ppm; MS (ES⁺) 564.2.

Step 9:5-(5-Isopropylsulfonyl-2-pyridyl)-3-[3-[4-(methylaminomethyl)phenyl]isoxazol-5-yl]pyridin-2-amine

TFA (1 mL, 12.98 mmol) was added to a stirred suspension of tert-butylN-[5-(5-isopropylsulfonyl-2-pyridyl)-3-[3-[4-(methylaminomethyl)phenyl]isoxazol-5-yl]-2-pyridyl]carbamate(105 mg, 0.1863 mmol) in DCM (5 mL) and the reaction stirred at ambienttemperature overnight. The solvent was removed in vacuo and the residueazeotroped with DCM (×2) and ether (×2). The reaction mixture was passedthrough a 2 g SCX-2 cartridge and washed with MeOH/DCM. The product waseluted by washing the cartridge with 2M NH₃ in MeOH. The fractions werecollected, concentrated in vacuo and the residue freeze-dried to givethe title compound as a cream solid (30 mg, 35% Yield). NMR (400.0 DMSO)δ 1.22 (d, J=6.9 Hz, 6H), 2.30 (s, 3H), 3.56 (sept, 1H), 3.73 (s, 2H),7.06 (br s, 2H), 7.51 (d, 2H), 7.63 (s, 1H), 7.93 (d, 2H), 8.24-8.31 (m,2H), 8.74 (d, 1H), 8.99 (dd, 1H) and 9.02 (d, 1H) ppm; MS (ES⁺) 464.1.

Example 85-(5-Isopropylsulfonyl-2-pyridyl)-3-[3-(p-tolyl)isoxazol-5-yl]pyridin-2-amine(Compound III-6)

Method H Step 1:5-(5-Isopropylsulfonyl-2-pyridyl)-3-[3-(p-tolyl)isoxazol-5-yl]pyridin-2-amine

TFA (1 mL, 12.98 mmol) was added to a stirred solution of tert-butylN-tert-butoxycarbonyl-N-[5-(5-isopropylsulfonyl-2-pyridyl)-3-[3-(p-tolyl)isoxazol-5-yl]-2-pyridyl]carbamate(90 mg, 0.1418 mmol) in DCM (5 mL) and the reaction stirred at ambienttemperature for 16 hours. The solvent was removed in vacuo and theresidue azeotroped with DCM (×2) and ether (×2). The residue wasredissolved in MeCN/water and passed through a sodium bicarbonatecartridge and freeze-dried to give the title compound as a pale yellowsolid (26 mg, 39% Yield). ¹H NMR (400.0 MHz, DMSO) δ 1.22 (d, J=6.8 Hz,6H), 2.40 (s, 3H), 3.56 (sept, 1H), 7.05 (s, 2H), 7.38 (d, J=8.0 Hz,2H), 7.61 (s, 1H), 7.88 (d, J=8.1 Hz, 2H), 8.24-8.31 (m, 2H), 8.74 (d,J=2.2 Hz, 1H), 8.99 (d, 1H) and 9.02 (d, 1H) ppm; MS (ES⁺) 435.1.

Example 93-[3-[2-Fluoro-4-(methylaminomethyl)phenyl]isoxazol-5-yl]-5-(5-isopropylsulfonyl-2-pyridyl)pyridin-2-amine(Compound III-8)

Method I Step 1: tert-Butyl(5-bromo-3-(3-(4-(((tert-butoxycarbonyl)(methyl)amino)methyl)-2-fluorophenyl)isoxazol-5-yl)pyridin-2-yl)(tert-butoxycarbonyl)carbamate

NCS (62.52 mg, 0.4682 mmol) was added to a solution tert-butyl3-fluoro-4-((hydroxyimino)methyl)benzyl(methyl)carbamate [prepared in ananalogous manner to that described in Bioorganic & Medicinal Chemistry,17(13), 4773-4785; 2009] (132.2 mg, 0.4682 mmol) in DMF (930.0 μL) andthe mixture heated at 55° C. for 15 minutes. The reaction mixture wasallowed to cool to room temperature, and tert-butylN-(5-bromo-3-ethynyl-2-pyridyl)-N-tert-butoxycarbonyl-carbamate (186 mg,0.4682 mmol) and Et₃N (56.85 mg, 78.31 μL, 0.5618 mmol) were added. Themixture was stirred at ambient temperature for 45 minutes followed byheating to 65° C. for 1 hour. The mixture was cooled to ambienttemperature and diluted with ethyl acetate (5 mL) and water (5 mL) andthe layers separated. The aqueous layer was extracted further with ethylacetate (2×5 mL) and the combined organic extracts washed with water(3×10 mL), dried over MgSO₄ and concentrated in vacuo. The residue waspurified by column chromatography (ISCO Companion™, 12 g column, elutingwith 0 to 100% EtOAc/Petroleum Ether, loaded in DCM) to give the impuresub-title product as a tacky yellow oil. This was used in the next stepwithout further purification.

Step 2: tert-ButylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-pyridyl]isoxazol-3-yl]-3-fluoro-phenyl]methyl]-N-methyl-carbamate

A mixture of tert-butylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-bromo-3-pyridyl]isoxazol-3-yl]-3-fluoro-phenyl]methyl]-N-methyl-carbamate(381.3 mg, 0.4615 mmol), bis(pinacolato)diboron (175.8 mg, 0.6922 mmol)and KOAc (135.8 mg, 1.384 mmol) in dioxane (6.626 mL) was degassed andplaced under an atmosphere of nitrogen. PdCl₂(dppf).DCM (37.69 mg,0.04615 mmol) added in one portion and the reaction mixture heated at95° C. for 1.5 hours. The reaction was cooled to ambient temperature anddiluted with EtOAc/brine. The layers were separated and the aqueouslayer extracted with EtOAc (×3). The combined organic extracts weredried (MgSO₄), filtered and concentrated in vacuo. The residue waspassed through a pad of fluorosil loading with DCM and eluting with0-50% EtOAc/Petroleum Ether. The relevant fractions were combined andconcentrated in vacuo to give the impure sub-title compound that wasused without further purification.

Step 3: tert-butylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-(5-isopropylsulfonyl-2-pyridyl)-3-pyridyl]isoxazol-3-yl]-3-fluoro-phenyl]methyl]-N-methyl-carbamate

A mixture of tert-butylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3-pyridyl]isoxazol-3-yl]-3-fluoro-phenyl]methyl]-N-methyl-carbamate(322.6 mg, 0.3339 mmol) and 2-bromo-5-isopropylsulfonyl-pyridine (105.8mg, 0.4007 mmol) in DMF (3 mL) was degassed and placed under anatmosphere of nitrogen. Pd(PPh₃)₂Cl₂ (13.81 mg, 0.03339 mmol) was addedand the reaction again degassed. 2M aqueous Na₂CO₃ (501.0 μL, 1.002mmol) was added and the reaction was stirred at 95° C. for 2 hours. Thereaction mixture was partitioned between EtOAc and brine and the aqueouslayer extracted with EtOAc (×3). The combined organic extracts werewashed with water (×3) and brine (×2), dried (MgSO₄), filtered andconcentrated in vacuo. The residue was purified by column chromatography(ISCO Companion™, 40 g column, loading with DCM and eluting with 0 to50% EtOAc/Petroleum Ether) to give the impure sub-title compound as ayellow oil which was used in the next step without purification.

Step 4:3-[3-[2-Fluoro-4-(methylaminomethyl)phenyl]isoxazol-5-yl]-5-(5-isopropylsulfonyl-2-pyridyl)pyridin-2-amine

TFA (1.724 g, 1.165 mL, 15.12 mmol) was added to a stirred solution oftert-butylN-[[4-[5-[2-[bis(tert-butoxycarbonyl)amino]-5-(5-isopropylsulfonyl-2-pyridyl)-3-pyridyl]isoxazol-3-yl]-3-fluoro-phenyl]methyl]-N-methyl-carbamate(198.7 mg, 0.1652 mmol) in DCM (7 mL) and the reaction stirred atambient temperature for 16 hours. The solvent was removed in vacuo andthe residue azeotroped with DCM (×2) and ether (×2). The material waspurified by reverse phase preparative HPLC [Waters Sunfire C18, 10 μM,100 Å column, gradient 10%-95% B (solvent A: 0.05% TFA in water; solventB: CH₃CN) over 16 minutes at 25 mL/min]. The fractions were collected,passed through a sodium bicarbonate cartridge and freeze-dried to givethe title compound as a white solid (6 mg, 7% Yield). 1H NMR (400.0 MHz,DMSO) δ 1.22 (d, J=6.8 Hz, 6H), 2.20-2.30 (m, 3H), 3.50-3.60 (m, 1H),3.75 (s, 2H), 7.05 (br s, 2H), 7.35-7.45 (m, 2H), 7.50 (s, 1H),7.90-8.00 (m, 1H), 8.25-8.40 (m, 2H) and 8.80 (s, 1H) ppm; MS (ES⁺)482.0.

Analytical Data

LCMS Exact Retention Cmpd Mass Time No (ES+) (min) HNMR III-1  492.10.58 H NMR (400.0 MHz, DMSO) d 2.10-2.20 (m, 2H), 2.25 (s, 1H), 2.29 (s,2H), 3.60-3.90 (m, 5H), 4.00-4.10 (m, 1H), 4.20-4.30 (m, 1H), 7.45 (d,2H), 7.50 (br s, 2H), 7.99 (d, J = 8.4 Hz, 2H), 8.08 (s, 2H), 8.20 (d, J= 8.3 Hz, 2H), 8.70 (s, 1H) and 8.75 (s, 1H) ppm III-2  441.2 3 H NMR(400.0 MHz, DMSO) d 1.69 (s, 1H), 1.79 (s, 1H), 2.68 (s, 1H), 2.79- 2.86(m, 2H), 2.98 (t, J = 4.9 Hz, 1H), 3.42 (dd, J = 5.9, 12.8 Hz, 2H), 3.67(d, J = 5.3 Hz, 2H), 7.50 (dd, J = 3.4, 7.9 Hz, 2H), 7.56 (s, 2H),7.63-7.71 (m, 3H), 7.84 (q, J = 3.9 Hz, 2H), 8.25-8.27 (m, 3H), 8.59 (d,J = 2.3 Hz, 1H) and 8.66 (d, J = 2.2 Hz, 1H) ppm III-3  392.9 2.94 H NMR(400.0 MHz, DMSO) d 3.27 (s, 3H), 7.64-7.69 (m, 5H), 8.01 (d, J = 8.5Hz, 2H), 8.07 (d, J = 8.6 Hz, 2H), 8.25- 8.27 (m, 2H), 8.66 (d, J = 2.5Hz, 1H) and 8.72 (d, J = 2.5 Hz, 1H) ppm III-4  411.95 3.52 H NMR (400.0MHz, DMSO) d 1.25 (t, 3H), 2.67 (br s, 2H), 3.16 (br s, 2H), 3.47 (br s,2H), 3.94 (br s, 2H), 6.25 (br s, 1H), 7.44 (s, 2H), 7.64-7.69 (m, 3H),8.22-8.24 (m, 2H), 8.29 (s, 1H) and 8.39 (s, 1H) ppm III-5  466.1 0.6 HNMR (400.0 MHz, DMSO) d 1.09 (d, J = 6.8 Hz, 6H), 2.12 (s, 3H), 3.58 (s,1H), 3.60-3.68 (m, 1H), 3.70 (s, 2H), 7.51 (d, J = 8.2 Hz, 2H), 7.99 (d,J = 8.2 Hz, 2H), 9.00 (m, 1H), 9.20 (m, 2H) and 9.51 (d, J = 1.3 Hz, 1H)ppm III-6  435.1 0.92 H NMR (400.0 MHz, DMSO) d 1.22 (d, J = 6.8 Hz,6H), 2.40 (s, 3H), 3.56 (sept, 1H), 7.05 (s, 2H), 7.38 (d, J = 8.0 Hz,2H), 7.61 (s, 1H), 7.88 (d, J = 8.1 Hz, 2H), 8.24-8.31 (m, 2H), 8.74 (d,J = 2.2 Hz, 1H), 8.99 (d, 1H) and 9.02 (d, 1H) ppm III-7  290.7 2.43H-CD3CN 9.5 bs (2H). 9.15 s (1H), 9.07 s (1H), 8.83 d (1H), 8.65 m (2H),8.4 s (1H), 8.0 m (1H), 7.7 m (2H), 7.45 m (2H), 7.25 m (1H) III-8  4820.63 H NMR (400.0 MHz, DMSO) d 1.22 (d, J = 6.8 Hz, 6H), 2.20-2.30 (m,3H), 3.50-3.60 (m, 1H), 3.75 (s, 2H), 7.05 (br s, 2H), 7.35-7.45 (m,2H), 7.50 (s, 1H), 7.90-8.00 (m, 1H), 8.25-8.40 (m, 2H) and 8.80 (s, 1H)ppm III-9  364.2 0.67 H NMR (400.0 MHz, DMSO) d 2.62- 2.68 (m, 3H), 4.28(t, J = 5.9 Hz, 2H), 7.51 (s, 1H), 7.69-7.76 (m, 4H), 7.97 (t, J = 2.1Hz, 1H), 8.35 (d, J = 8.3 Hz, 2H), 8.59 (d, J = 2.4 Hz, 1H), 8.70 (d, J= 2.4 Hz, 1H) and 8.89 (s, 2H) ppm III-10 465.3 0.65 H NMR (400.0 MHz,DMSO) d 1.23 (d, J = 6.8 Hz, 6H), 2.30 (s, 3H), 3.56 (sept, 1H), 3.77(s, 2H), 7.60 (d, 2H), 7.91 (s, 2H), 8.16 (d, 2H), 8.29 (dd, 1H), 8.36(d, 1H), 9.00 (d, 1H), 9.03 (d, 1H) and 9.12 (d, 1H) ppm III-11 464.30.6 H NMR (400.0 MHz, DMSO) d 1.20 (d, J = 6.8 Hz, 6H), 2.54 (s, 3H),3.48- 3.50 (m, 1H), 4.13 (s, 2H), 7.70 (d, J = 8.2 Hz, 3H), 7.93 (d, J =7.7 Hz, 3H), 8.08 (d, J = 8.5 Hz, 2H), 8.31 (d, J = 8.3 Hz, 2H), 8.68(d, J = 2.5 Hz, 1H) and 8.74 (d, J = 2.5 Hz, 1H) ppm III-12 464.1 0.62 HNMR (400.0 MHz, DMSO) d 1.22 (d, J = 6.9 Hz, 6H), 2.30 (s, 3H), 3.56(sept, 1H), 3.73 (s, 2H), 7.06 (br s, 2H), 7.51 (d, 2H), 7.63 (s, 1H),7.93 (d, 2H), 8.24-8.31 (m, 2H), 8.74 (d, 1H), 8.99 (dd, 1H) and 9.02(d, 1H) ppm

Examples 10 Cellular ATR Inhibition Assay

Compounds can be screened for their ability to inhibit intracellular ATRusing an immunofluorescence microscopy assay to detect phosphorylationof the ATR substrate histone H2AX in hydroxyurea treated cells. HT29cells are plated at 14,000 cells per well in 96-well black imagingplates (BD 353219) in McCoy's 5A media (Sigma M8403) supplemented with10% foetal bovine serum (JRH Biosciences 12003), Penicillin/Streptomycinsolution diluted 1:100 (Sigma P7539), and 2 mM L-glumtamine (SigmaG7513), and allowed to adhere overnight at 37° C. in 5% CO₂. Compoundsare then added to the cell media from a final concentration of 25 μM in3-fold serial dilutions and the cells are incubated at 37° C. in 5% CO₂.After 15 min, hydroxyurea (Sigma H8627) is added to a finalconcentration of 2 mM.

After 45 min of treatment with hydroxyurea, the cells are washed in PBS,fixed for 10 min in 4% formaldehyde diluted in PBS (Polysciences Inc18814), washed in 0.2% Tween-20 in PBS (wash buffer), and permeabilisedfor 10 min in 0.5% Triton X-100 in PBS, all at room temperature. Thecells are then washed once in wash buffer and blocked for 30 min at roomtemperature in 10% goat serum (Sigma G9023) diluted in wash buffer(block buffer). To detect H2AX phosphorylation levels, the cells arethen incubated for 1 h at room temperature in primary antibody (mousemonoclonal anti-phosphorylated histone H2AX Ser139 antibody; Upstate05-636) diluted 1:250 in block buffer. The cells are then washed fivetimes in wash buffer before incubation for 1 h at room temperature inthe dark in a mixture of secondary antibody (goat anti-mouse Alexa Fluor488 conjugated antibody; Invitrogen A11029) and Hoechst stain(Invitrogen H3570); diluted 1:500 and 1:5000, respectively, in washbuffer. The cells are then washed five times in wash buffer and finally100 ul PBS is added to each well before imaging.

Cells are imaged for Alexa Fluor 488 and Hoechst intensity using the BDPathway 855 Bioimager and Attovision software (BD Biosciences, Version1.6/855) to quantify phosphorylated H2AX Ser139 and DNA staining,respectively. The percentage of phosphorylated H2AX-positive nuclei in amontage of 9 images at 20× magnification is then calculated for eachwell using BD Image Data Explorer software (BD Biosciences Version2.2.15). Phosphorylated H2AX-positive nuclei are defined asHoechst-positive regions of interest containing Alexa Fluor 488intensity at 1.75-fold the average Alexa Fluor 488 intensity in cellsnot treated with hydroxyurea. The percentage of H2AX positive nuclei isfinally plotted against concentration for each compound and IC50s forintracellular ATR inhibition are determined using Prism software(GraphPad Prism version 3.0cx for Macintosh, GraphPad Software, SanDiego Calif., USA).

The compounds described herein can also be tested according to othermethods known in the art (see Sarkaria et al, “Inhibition of ATM and ATRKinase Activities by the Radiosensitizing Agent, Caffeine: CancerResearch 59: 4375-5382 (1999); Hickson et al, “Identification andCharacterization of a Novel and Specific Inhibitor of theAtaxia-Telangiectasia Mutated Kinase ATM” Cancer Research 64: 9152-9159(2004); Kim et al, “Substrate Specificities and Identification ofPutative Substrates of ATM Kinase Family Members” The Journal ofBiological Chemistry, 274(53): 37538-37543 (1999); and Chiang et al,“Determination of the catalytic activities of mTOR and other members ofthe phosphoinositide-3-kinase-related kinase family” Methods Mol. Biol.281:125-41 (2004)).

Example 11 ATR Inhibition Assay

Compounds were screened for their ability to inhibit ATR kinase using aradioactive-phosphate incorporation assay. Assays were carried out in amixture of 50 mM Tris/HCl (pH 7.5), 10 mM MgCl₂ and 1 mM DTT. Finalsubstrate concentrations were 10 μM [γ-33P]ATP (3mCi 33P ATP/mmol ATP,Perkin Elmer) and 800 μM target peptide (ASELPASQPQPFSAKKK).

Assays were carried out at 25° C. in the presence of 5 nM full-lengthATR. An assay stock buffer solution was prepared containing all of thereagents listed above, with the exception of ATP and the test compoundof interest. 13.5 μL of the stock solution was placed in a 96 well platefollowed by addition of 2 μL of DMSO stock containing serial dilutionsof the test compound (typically starting from a final concentration of15 μM with 3-fold serial dilutions) in duplicate (final DMSOconcentration 7%). The plate was pre-incubated for 10 minutes at 25° C.and the reaction initiated by addition of 15 μL [γ-33P]ATP (finalconcentration 10 μM).

The reaction was stopped after 24 hours by the addition of 30 μL 0.1Mphosphoric acid containing 2 mM ATP. A multiscreen phosphocellulosefilter 96-well plate (Millipore, Cat no. MAPHN0B50) was pretreated with100 μL 0.2M phosphoric acid prior to the addition of 45 μL of thestopped assay mixture. The plate was washed with 5×200 μL 0.2Mphosphoric acid. After drying, 100 μL Optiphase ‘SuperMix’ liquidscintillation cocktail (Perkin Elmer) was added to the well prior toscintillation counting (1450 Microbeta Liquid Scintillation Counter,Wallac).

After removing mean background values for all of the data points,Ki(app) data were calculated from non-linear regression analysis of theinitial rate data using the Prism software package (GraphPad Prismversion 3.0cx for Macintosh, GraphPad Software, San Diego Calif., USA).

Below is a chart showing the ATR Inhibition Ki values of compounds ofthe disclosure. Compounds with a Ki value of ≦10 nM are marked with“+++.” Compounds with a Ki value >10 nM but ≦100 nM are marked with“++.” Compounds with a Ki value >100 nM but ≦5 uM are marked with “+.”

Compound No. Ki Value III-1 +++ III-2 ++ III-3 ++ III-4 ++ III-5 +++III-6 ++ III-7 + III-8 +++ III-9 ++  III-10 +++  III-11 +++  III-12 +++

Example 12 Cisplatin Sensitization Assay

Compounds can be screened for their ability to sensitize HCT116colorectal cancer cells to Cisplatin using a 96 h cell viability (MTS)assay. HCT116 cells, which possess a defect in ATM signaling toCisplatin (see, Kim et al.; Oncogene 21:3864 (2002); see also, Takemuraet al.; JBC 281:30814 (2006)) are plated at 470 cells per well in96-well polystyrene plates (Costar 3596) in 150 μl of McCoy's 5A media(Sigma M8403) supplemented with 10% foetal bovine serum (JRH Biosciences12003), Penicillin/Streptomycin solution diluted 1:100 (Sigma P7539),and 2 mM L-glumtamine (Sigma G7513), and allowed to adhere overnight at37° C. in 5% CO₂. Compounds and Cisplatin are then both addedsimultaneously to the cell media in 2-fold serial dilutions from a topfinal concentration of 10 μM as a full matrix of concentrations in afinal cell volume of 200 μl, and the cells are then incubated at 37° C.in 5% CO₂. After 96 h, 40 μl of MTS reagent (Promega G358a) is added toeach well and the cells are incubated for 1 h at 37° C. in 5% CO₂.Finally, absorbance is measured at 490 nm using a SpectraMax Plus 384reader (Molecular Devices) and the concentration of compound required toreduce the IC50 of Cisplatin alone by at least 3-fold (to 1 decimalplace) can be reported (CP3 shift).

Cmpd No. CP3 Shift (uM) III-8  .234 III-10 .078 III-11 .156 III-12 .156

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds, methods, and processes of thisinvention. Therefore, it will be appreciated that the scope of thisinvention is to be defined by the appended claims rather than by thespecific embodiments that have been represented by way of exampleherein.

1. A compound of formula III:

or a pharmaceutically acceptable salt thereof, wherein L₂ is C₁₋₁₀aliphatic chain where up to 3 methylene units of said chain areoptionally replaced with —O—, —S—, —N(R′)—, or —CO—; R′ is H orC₁₋₄alkyl; m is 0 or 1; R² is -Q or -Q-Q¹; Q is a 3-8 memberedmonocyclic ring having 0-4 heteroatoms independently selected fromnitrogen, oxygen, or sulfur; each Q is independently and optionallysubstituted with 1-4 J^(Q) groups; Q is optionally fused to Q¹ to form afused bicyclic ring Q-Q¹; or Q and Q¹ are optionally joined together ata carbon atom to form a spirocyclic bicyclic ring Q-Q¹; or Q and Q¹,taken together, form a bridged bicyclic ring Q-Q¹ wherein said bridge is1-3 atoms long; Q¹ is a 3-8 membered monocyclic ring having 0-4heteroatoms independently selected from nitrogen, oxygen, or sulfur;each Q¹ is independently and optionally substituted with 1-4 J^(Q1)groups; L is —C(O)NH— or —C(O)N(C₁₋₆alkyl)-; n is 0 or 1; R¹ is a 5-6membered monocyclic aryl or heteroaryl ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; wherein saidmonocyclic aryl or heteroaryl ring is optionally fused to another ringto form a 8-10 membered bicyclic aryl or heteroaryl ring having 0-6heteroatoms independently selected from nitrogen, oxygen, or sulfur; R¹is optionally substituted with 1-5 J¹ groups; each J^(Q), J^(Q1), and J¹is independently halo, —CN, —NO₂, V—R, or —(V²)_(m)-Q³; V is aC₁₋₁₀aliphatic chain wherein 0-3 methylene units are optionally andindependently replaced with oxygen, nitrogen, sulfur, C(O), S(O), orS(O)₂; V is optionally substituted with 1-6 occurrences of J^(V); V² isa C₁₋₁₀aliphatic chain wherein 0-3 methylene units are optionally andindependently replaced with oxygen, nitrogen, sulfur, C(O), S(O), orS(O)₂; V is optionally substituted with 1-6 occurrences of J^(V2); m is0 or 1; Q³ is a 3-8 membered saturated or unsaturated monocyclic ringhaving 0-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur, or a 8-10 membered saturated or unsaturated bicyclic ring having0-6 heteroatoms independently selected from nitrogen, oxygen, or sulfur;each Q³ is optionally substituted with 1-5 J^(Q3); each J^(V) and J^(V2)is independently halogen, CN, NH₂, NO₂, C₁₋₄aliphatic,NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, OH, O(C₁₋₄aliphatic), CO₂H,CO(C₁₋₄aliphatic), CO₂(C₁₋₄aliphatic), C(O)NH₂, C(O)NH(C₁₋₄aliphatic),C(O)N(C₁₋₄aliphatic)₂, —NHCO(C₁₋₄aliphatic),N(C₁₋₄aliphatic)CO(C₁₋₄aliphatic), SO₂(C₁₋₄aliphatic),NHSO₂(C₁₋₄aliphatic), or N(C₁₋₄aliphatic)SO₂(C₁₋₄aliphatic), whereinsaid C₁₋₄aliphatic is optionally substituted with halo; J^(Q3) is halo,oxo, CN, NO₂, X—R, or —(X)_(p)-Q⁴, p is 0 or 1; X is C₁₋₄aliphatic;wherein 1-3 methylene units of said C₁₋₆aliphatic are optionallyreplaced with —NR, —O—, —S—, C(O), S(O)₂, or S(O); wherein X isoptionally and independently substituted with 1-4 occurrences of NH₂,NH(C₁₋₄aliphatic), N(C₁₋₄aliphatic)₂, halogen, C₁₋₄aliphatic, OH,O(C₁₋₄aliphatic), NO₂, CN, CO(C₁₋₄aliphatic), CO₂H, CO₂(C₁₋₄aliphatic),C(O)NH₂, C(O)NH(C₁₋₄aliphatic), C(O)N(C₁₋₄aliphatic)₂,SO(C₁₋₄aliphatic), SO₂(C₁₋₄aliphatic), SO₂NH(C₁₋₄aliphatic),SO₂NH(C₁₋₄aliphatic), NHC(O)(C₁₋₄aliphatic),N(C₁₋₄aliphatic)C(O)(C₁₋₄aliphatic), NHSO₂(C₁₋₄aliphatic), orN(C₁₋₄aliphatic)SO₂(C₁₋₄aliphatic), wherein said C₁₋₄aliphatic isoptionally substituted with 1-3 occurrences of halo; Q⁴ is a 3-8membered saturated or unsaturated monocyclic ring having 0-4 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, or a 8-10membered saturated or unsaturated bicyclic ring having 0-6 heteroatomsindependently selected from nitrogen, oxygen, or sulfur; each Q⁴ isoptionally substituted with 1-5 J^(Q4); each J^(V), J^(V2), and J^(Q4)is independently halo, CN, or C₁₋₄alkyl wherein up to 2 methylene unitsare optionally replaced with O, N, S, C(O), S(O), or S(O)₂; R is H orC₁₋₄alkyl wherein said C₁₋₄alkyl is optionally substituted with 1-4halo.
 2. The compound of claim 1, wherein m is
 0. 3. The compound ofclaim 2, wherein n is
 0. 4. The compound of claim 3, wherein R¹ isisoxazolyl or oxadiazolyl.
 5. The compound of claim 4, wherein R¹ isoptionally substituted with one occurrence of J¹.
 6. The compound ofclaim 5, wherein J¹ is —(V²)_(m)-Q³ wherein m is
 0. 7. The compound ofclaim 6, wherein J¹ is phenyl.
 8. The compound of claim 7, whereinJ^(Q3) is halo or X—R.
 9. The compound of claim 5, wherein J¹ is phenyloptionally substituted with CH₂NHCH₃, methyl, or fluoro.
 10. Thecompound of claim 1, wherein n is
 1. 11. The compound of claim 10,wherein L is —C(O)NH— and R¹ is phenyl.
 12. The compound of claim 2,wherein Q is aromatic.
 13. The compound of claim 12, wherein R² isphenyl, pyridinyl, pyrimidinyl, pyrazinyl, or thienyl.
 14. The compoundof claim 13, wherein R² is pyridinyl.
 15. The compound of claim 1,wherein Q is nonaromatic.
 16. The compound of claim 15, wherein R² is1,2,3,6-tetrahydropyridinyl or N-alkyl pyridone.
 17. The compound ofclaim 1, wherein J^(Q) is V—R or —(V²)_(m)-Q³.
 18. The compound of claim17, wherein V is S(O)₂ or C(O); and V² is S(O)₂ or C(O).
 19. Thecompound of claim 18, wherein R is C₁₋₄alkyl and Q³ is a 3-7 memberedsaturated or unsaturated monocyclic ring having 0-2 heteroatomsindependently selected from nitrogen, oxygen, or sulfur.
 20. Thecompound of claim 1, wherein n is 0 or 1; L is —C(O)NH—; R¹ is phenyl,oxadiazolyl, or isoxazolyl; m is 0; R² is phenyl, pyridinyl,pyrimidinyl, pyrazinyl, thienyl, or 1,2,3,6-tetrahydropyridinyl; andJ^(Q) is V—R or —(V²)_(m)-Q³ wherein V is SO₂ or CO, R is C₁₋₄alkyl; V²is S(O)₂ or C(O); and Q³ is a 5-7 membered monocyclic ring having 0-2heteroatoms selected from oxygen, nitrogen, or sulfur.
 21. The compoundof claim 1, wherein n is 0 or 1; L is —C(O)NH—; R¹ is phenyl,oxadiazolyl, or isoxazolyl; J¹ is phenyl optionally substituted withCH₂NHCH₃, methyl, or fluoro; m is 0; R² is phenyl, pyridinyl, pyrazinyl,thienyl, or 1,2,3,6-tetrahydropyridinyl; and J^(Q) is SO₂(furanyl),CO(homopiperazinyl), or SO₂(C₁₋₆alkyl).
 22. The compound of claim 1selected from the following:


23. A pharmaceutical composition comprising a compound of claim 1 and apharmaceutically acceptable carrier.
 24. A method for treating cancer ina patient comprising administering a compound of claim 1 or apharmaceutically acceptable derivative thereof.
 25. The method of claim24, further comprising administering to said patient an additionaltherapeutic agent selected from a DNA-damaging agent; wherein saidadditional therapeutic agent is appropriate for the disease beingtreated; and said additional therapeutic agent is administered togetherwith said compound as a single dosage form or separately from saidcompound as part of a multiple dosage form.
 26. The method of claim 25,wherein said DNA-damaging agent is selected chemotherapy or radiationtreatment.
 27. The method of claim 25, wherein said DNA-damaging agentis selected from ionizing radiation, radiomimetic neocarzinostatin, aplatinating agent, a Topo I inhibitor, a Topo II inhibitor, anantimetabolite, an alkylating agent, an alkyl sulphonates, anantimetabolite, or an antibiotic.
 28. The method of claim 27, whereinsaid DNA-damaging agent is selected from ionizing radiation, aplatinating agent, a Topo I inhibitor, a Topo II inhibitor, or anantibiotic.
 29. The method of claim 28, wherein said platinating agentis selected from Cisplatin, Oxaliplatin, Carboplatin, Nedaplatin,Lobaplatin, Triplatin Tetranitrate, Picoplatin, Satraplatin, ProLindacand Aroplatin; said Topo I inhibitor is selected from Camptothecin,Topotecan, Irinotecan/SN38, Rubitecan and Belotecan; said Topo IIinhibitor is selected from Etoposide, Daunorubicin, Doxorubicin,Aclarubicin, Epirubicin, Idarubicin, Amrubicin, Pirarubicin, Valrubicin,Zorubicin and Teniposide; said antimetabolite is selected fromAminopterin, Methotrexate, Pemetrexed, Raltitrexed, Pentostatin,Cladribine, Clofarabine, Fludarabine, Thioguanine, Mercaptopurine,Fluorouracil, Capecitabine, Tegafur, Carmofur, Floxuridine, Cytarabine,Gemcitabine, Azacitidine and Hydroxyurea; said alkylating agent isselected from Mechlorethamine, Cyclophosphamide, Ifosfamide,Trofosfamide, Chlorambucil, Melphalan, Prednimustine, Bendamustine,Uramustine, Estramustine, Carmustine, Lomustine, Semustine, Fotemustine,Nimustine, Ranimustine, Streptozocin, Busulfan, Mannosulfan, Treosulfan,Carboquone, ThioTEPA, Triaziquone, Triethylenemelamine, Procarbazine,Dacarbazine, Temozolomide, Altretamine, Mitobronitol, Actinomycin,Bleomycin, Mitomycin and Plicamycin.
 30. The method of claim 29, whereinsaid platinating agent is selected from Cisplatin, Oxaliplatin,Carboplatin, Nedaplatin, or Satraplatin; said Topo I inhibitor isselected from Camptothecin, Topotecan, irinotecan/SN38, rubitecan; saidTopo II inhibitor is selected from Etoposide; said antimetabolite isselected from methotrexate, pemetrexed, Thioguanine, Fludarabine,Cladribine, Cytarabine, gemcitabine, 6-Mercaptopurine, or5-Fluorouracil; said alkylating agent is selected from nitrogenmustards, nitrosoureas, triazenes, alkyl sulfonates, Procarbazine, oraziridines; and said antibiotic is selected from Hydroxyurea,Anthracyclines, Anthracenediones, or Streptomyces family.
 31. The methodof claim 28 wherein said DNA-damaging agent is a platinating agent orionizing radiation.
 32. The method of any one of claims 24-31, whereinsaid cancer is a solid tumor selected from the following cancers: Oral:buccal cavity, lip, tongue, mouth, pharynx; Cardiac: sarcoma(angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma), myxoma,rhabdomyoma, fibroma, lipoma and teratoma; Lung: bronchogenic carcinoma(squamous cell or epidermoid, undifferentiated small cell,undifferentiated large cell, adenocarcinoma), alveolar (bronchiolar)carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatoushamartoma, mesothelioma; Gastrointestinal: esophagus (squamous cellcarcinoma, larynx, adenocarcinoma, leiomyosarcoma, lymphoma), stomach(carcinoma, lymphoma, leiomyosarcoma), pancreas (ductal adenocarcinoma,insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma), smallbowel or small intestines (adenocarcinoma, lymphoma, carcinoid tumors,Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel or large intestines (adenocarcinoma, tubularadenoma, villous adenoma, hamartoma, leiomyoma), colon, colon-rectum,colorectal; rectum, Genitourinary tract: kidney (adenocarcinoma, Wilm'stumor [nephroblastoma], lymphoma), bladder and urethra (squamous cellcarcinoma, transitional cell carcinoma, adenocarcinoma), prostate(adenocarcinoma, sarcoma), testis (seminoma, teratoma, embryonalcarcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cellcarcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma); Liver:hepatoma (hepatocellular carcinoma), cholangiocarcinoma, hepatoblastoma,angiosarcoma, hepatocellular adenoma, hemangioma, biliary passages;Bone: osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumorchordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors; Nervous system: skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord neurofibroma, meningioma, glioma, sarcoma); Gynecological:uterus (endometrial carcinoma), cervix (cervical carcinoma, pre-tumorcervical dysplasia), ovaries (ovarian carcinoma [serouscystadenocarcinoma, mucinous cystadenocarcinoma, unclassifiedcarcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors,dysgerminoma, malignant teratoma), vulva (squamous cell carcinoma,intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma),vagina (clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma(embryonal rhabdomyosarcoma), fallopian tubes (carcinoma), breast; Skin:malignant melanoma, basal cell carcinoma, squamous cell carcinoma,Karposi's sarcoma, keratoacanthoma, moles dysplastic nevi, lipoma,angioma, dermatofibroma, keloids, psoriasis, Thyroid gland: papillarythyroid carcinoma, follicular thyroid carcinoma; undifferentiatedthyroid cancer, medullary thyroid carcinoma, multiple endocrineneoplasia type 2A, multiple endocrine neoplasia type 2B, familialmedullary thyroid cancer, pheochromocytoma, paraganglioma; and Adrenalglands: neuroblastoma.
 33. The method of claim 32, wherein said canceris selected from lung cancer, head and neck cancer, pancreatic cancer,gastric cancer, and brain cancer. 34-46. (canceled)